Flow cytometry electrotransfection device

Abstract

The utility model belongs to the technical field of medical instruments, and particularly relates to a flow cytometry electrotransfection device which comprises a first electrode plate, a second electrode plate and an electrotransfection cavity, an electric rotating cavity, an annular channel, a flow guide cavity, an inlet hole communicated with the electric rotating cavity and an outlet hole communicated with the flow guide cavity are formed in the electric rotating cavity, and the two opposite ends of the annular channel are communicated with the electric rotating cavity and the flow guide cavity respectively; the first electrode plate and the second electrode plate are respectively mounted on two opposite inner side walls of the electric rotating cavity; the axis of the electric rotating cavity, the axis of the annular channel and the center line of the inlet hole coincide. The cell electrotransfection device has the capability of continuously treating large-volume cell sap, and has no risk of channel blockage; meanwhile, the uniformity of cell electrotransfection results is also ensured, so that the overall transfection efficiency and motility are improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical devices, in particular to a flow cytometry electroporation device. BACKGROUND

[0002] Cell electroporation technology (Electroporation) is also known as cell electroporation technology, which is a typical non-biological cell transfection technology. It uses a relatively short time (a few milliseconds to a few tens of milliseconds) of high-intensity electric pulse to directly act on the cell membrane, so that a small hole (electroporation) is temporarily formed, allowing exogenous macromolecular substances (such as DNA, RNA, protein, etc.) to enter the cell through the hole. After a short time (a few milliseconds to a few seconds) of self-repair of the cell membrane, the hole is closed, thereby realizing cell electroporation.

[0003] Conventional cell electroporation usually uses two parallel plate electrodes for electroporation. By applying a short-time voltage on the two electrode sheets, an electric field is formed between the electrode sheets, and the cells in the region are subjected to electric field to realize the electroporation of exogenous substances. Although this method can achieve certain cell transfection, the voltage experienced by cells at different positions in the electric field is different, resulting in low overall transfection efficiency. In addition, the conventional electroporation device can only realize single electroporation and cannot process large volume solutions and continuous flow electroporation.

[0004] Therefore, researchers have improved it by building a micro-channel or micro-cavity between the two parallel plate electrodes to realize the continuous flow electroporation processing capacity (CN112639112A). Although this flow cytometry electroporation device can achieve a certain volume of continuous flow electroporation, the volume of the electroporation chamber is small, and the sample amount and flux processed by the continuous flow method are limited. In addition, during the relatively high-flow cell electroporation process, the fluid distribution in the electroporation cavity is uneven, especially in the horizontal direction perpendicular to the fluid flow direction, resulting in a significant difference in the time that cells at different positions are subjected to the electric field during the continuous flow process, which makes the overall cell transfection result uneven. SUMMARY

[0005] The present application aims to solve the technical problems in the prior art that cell electroporation cannot realize high-throughput large-volume cell electroporation and continuous flow electroporation, and provides a flow cytometry electroporation device.

[0006] To solve the above technical problems, the first embodiment of the present application provides a cell electroporation device, which comprises a first electrode sheet, a second electrode sheet and an electroporation cavity; the electroporation cavity is provided with an electroporation cavity, an annular channel, a flow guide cavity, an inlet hole communicating with the electroporation cavity and an outlet hole communicating with the flow guide cavity, and the opposite ends of the annular channel are respectively communicated with the electroporation cavity and the flow guide cavity;

[0007] The first electrode sheet and the second electrode sheet are respectively installed on opposite two inner walls of the electro-translational cavity;

[0008] The axis of the electro-translational cavity, the axis of the annular channel and the center line of the inlet hole coincide.

[0009] Optionally, the annular channel comprises a plurality of arc-shaped channels, the plurality of arc-shaped channels are annularly and spacedly distributed around the axis of the electro-translational cavity, and the central angle and the width of each arc-shaped channel are equal.

[0010] Optionally, the flow guide cavity is a circular truncated cone cavity, the center line of the inlet hole, the axis of the electro-translational cavity, the axis of the circular truncated cone cavity and the center line of the outlet hole coincide; the inner diameter of the circular truncated cone cavity gradually increases from the outlet hole to one end of the annular channel.

[0011] Optionally, the first electrode sheet comprises a first electrode circular sheet and a first electrode lead-out sheet connected to the first electrode circular sheet, and the first electrode circular sheet is attached to the top inner wall of the electro-translational cavity;

[0012] The second electrode sheet comprises a second electrode circular sheet and a second electrode lead-out sheet connected to the second electrode circular sheet, and the second electrode circular sheet is attached to the bottom inner wall of the electro-translational cavity;

[0013] The first electrode circular sheet and the second electrode circular sheet are arranged in parallel, the diameter of the first electrode circular sheet is equal to the diameter of the second electrode circular sheet, and the axis of the first electrode circular sheet, the axis of the second electrode circular sheet and the axis of the electro-translational cavity coincide.

[0014] Optionally, the electro-translational cavity body comprises a first cover plate, an electro-translational block, an intermediate block and a second cover plate which are sequentially and laminatedly installed; the inlet hole is arranged on the first cover plate, the electro-translational cavity is arranged on the electro-translational block, the annular channel is arranged on the intermediate block, and the flow guide cavity and the outlet hole are both arranged on the second cover plate.

[0015] Optionally, the first cover plate is further provided with a first accommodating groove communicating with the inlet hole, and the first electrode sheet is installed in the first accommodating groove;

[0016] The intermediate block is further provided with a second accommodating groove, the annular channel is arranged around the second accommodating groove, and the second electrode sheet is installed in the second accommodating groove.

[0017] Optionally, the first cover plate is further provided with a first annular positioning groove arranged around the first electrode sheet, and the electro-translational block is further provided with a first annular positioning protrusion arranged around the electro-translational cavity; the first annular positioning protrusion is inserted into the first annular positioning groove;

[0018] The end of the electric rotation block away from the first annular positioning protrusion is provided with a second annular positioning groove, the second annular positioning groove is arranged around the electric rotation cavity, the intermediate block is provided with a second annular positioning protrusion arranged around the annular channel; the second annular positioning protrusion is inserted into the second annular positioning groove;

[0019] The end of the intermediate block away from the second annular positioning protrusion is provided with a third annular positioning protrusion, the third annular positioning protrusion is arranged around the annular channel, and the second cover plate is further provided with a third annular positioning groove arranged around the flow guide cavity; the third annular positioning protrusion is inserted into the third annular positioning groove.

[0020] Optionally, the first cover plate is further provided with a first threaded hole, the first threaded hole is communicated with the end of the inlet hole away from the electric rotation cavity; and / or

[0021] The second cover plate is further provided with a second threaded hole, the second threaded hole is communicated with the end of the outlet hole away from the flow guide cavity.

[0022] Optionally, the first electrode sheet is further provided with a through hole, the inlet hole is communicated with the electric rotation cavity through the through hole.

[0023] Optionally, the electric rotation cavity is columnar.

[0024] In the application, the electric rotation cavity is provided with an inlet hole, an electric rotation cavity, an annular channel, a flow guide cavity and an outlet hole communicated in sequence, the axis of the electric rotation cavity coincides with the center line of the inlet hole; cell liquid flows into the electric rotation cavity through the inlet hole, the electric rotation cavity is a columnar hole or a circular truncated cone hole, the cell liquid vertically flows from the center position of the electric rotation cavity and then uniformly diverges in the radial direction of the electric rotation cavity (360 degrees), and the flow speed in each radial direction of the electric rotation cavity is the same. Specifically, the cell liquid flows into the electric rotation cavity from the narrow inlet hole, the inlet hole is relatively narrow, and the flow speed of the cell liquid in the inlet hole is relatively large; the space of the electric rotation cavity is larger, and the flow speed of the cell liquid at the inlet of the electric rotation cavity suddenly decreases, the flow direction is turned by 90 degrees and uniformly diverges, and the flow speed of the cell liquid at different radial positions far away from the inlet is almost unchanged; at the same time, before the cell liquid flows into the electric rotation cavity from the inlet hole, the flow direction of the cell liquid is along the axial direction of the electric rotation cavity, and with the flow of the cell liquid in the electric rotation cavity, the flow direction of the cell liquid changes to the radial direction of the electric rotation cavity, and the continuous flow speed at different horizontal positions is the same; since the flow type cell electric rotation device adopts an axisymmetric design, a plurality of coaxial cylindrical surfaces are taken around the axis of the electric rotation cavity as the center axis, and through software simulation analysis, the flow speed distribution and size of the cell liquid on each cylindrical surface remain the same.

[0025] In addition, the first electrode sheet and the second electrode sheet are respectively installed on opposite two inner side walls of the electrotransformation cavity; the first electrode sheet and the second electrode sheet are arranged in parallel, can form a uniformly distributed electric field in the electrotransformation cavity, and the electric field line direction is the axial direction of the electrotransformation cavity, and the electric field line direction is perpendicular to the flow direction of the cell liquid in the electrotransformation cavity, so that the electric field intensity of the cells at different positions in the electrotransformation cavity at the same time can be ensured to be consistent, and the uniformity and stability of cell transfection in the electrotransformation cavity are improved.

[0026] In the present application, the cell liquid flows in the radial direction in the electrotransformation cavity, and due to the axisymmetric design, the flow of the cell liquid in each direction on the same cylindrical surface is equal, and the electric field perpendicular to the flow direction of the cell liquid can be formed in the electrotransformation cavity, so that the cell liquid can be stably and uniformly transfection in the electrotransformation cavity; after the cell liquid is transfection in the electrotransformation cavity, the cell liquid enters the flow guide cavity from the annular channel, and since the axis of the flow guide cavity coincides with the axis of the electrotransformation cavity, the flux of the cell liquid in each direction of the annular channel is equal; the cell liquid converges in the flow guide cavity and is then discharged through the outlet hole.

[0027] In the present application, the cell electrotransfection device has the ability to continuously process large volume of cell liquid, supports long time and high flux electrotransfection of cells, and has no risk of channel blockage. At the same time, the highly uniform electric field distribution in the electrotransformation cavity and the symmetric flow field design can ensure that each cell in the continuous flow experiences the same electric field intensity, the speed of the continuous flow of the cell liquid in the electrotransformation cavity in the radial direction and at different heights is the same, and the flow direction is consistent. The flow direction of the uniform flow is perpendicular to the direction of the uniform electric field, so that each cell in the continuous flow experiences the same number of times and time of electric field shock, ensuring the uniformity of the cell electrotransfection result, thereby improving the overall transfection efficiency and viability. BRIEF DESCRIPTION OF DRAWINGS

[0028] The present application will be further described below in conjunction with the drawings and examples.

[0029] Figure 1 is a structural schematic view of a cell electrotransfection device provided by an embodiment of the present application;

[0030] Figure 2 is a sectional view of a cell electrotransfection device provided by an embodiment of the present application;

[0031] Figure 3 is a structural schematic view of a first electrode sheet of a cell electrotransfection device provided by an embodiment of the present application installed on a first cover plate;

[0032] Figure 4 is a structural schematic view of an electrotransformation block of a cell electrotransfection device provided by an embodiment of the present application;

[0033] Figure 5 Figure 2 is a schematic diagram of the structure of the second electrode sheet of the cell electroporation device according to an embodiment of the present application;

[0034] Figure 6 Figure 2 is a schematic diagram of the structure of the second electrode sheet of the cell electroporation device according to an embodiment of the present application;

[0035] Figure 7 Figure 3 is a schematic diagram of the axial section of the electroporation cavity and the flow of the cell solution in the electroporation cavity according to an embodiment of the present application;

[0036] Figure 8 Figure 3 is a schematic diagram of the axial section of the electroporation cavity and the flow of the cell solution in the electroporation cavity according to an embodiment of the present application;

[0037] Figure 9 Figure 4 is a schematic diagram of the horizontal section of the fluid velocity and flow direction distribution of the cell flow into the electroporation cavity according to an embodiment of the present application;

[0038] Figure 10 Figure 5 is a schematic diagram of the fluid velocity distribution of the cell flow into the electroporation cavity at three different locations according to an embodiment of the present application;

[0039] Figure 11 Figure 6 is a schematic diagram of the uniform electric field distribution and electric field direction in the electroporation cavity according to an embodiment of the present application.

[0040] The reference signs in the specification are as follows:

[0041] 1, first electrode sheet; 11, first electrode disc; 12, first electrode lead-out sheet; 13, through hole; 2, second electrode sheet; 21, second electrode disc; 22, second electrode lead-out sheet; 3, electroporation cavity; 31, electroporation cavity; 32, annular channel; 321, arc-shaped channel; 33, flow guide cavity; 34, inlet hole; 35, outlet hole; 36, first cover plate; 361, first accommodating groove; 362, first annular positioning groove; 363, first threaded hole; 37, electroporation block; 371, first annular positioning protrusion; 372, second annular positioning groove; 38, intermediate block; 381, second accommodating groove; 382, second annular positioning protrusion; 383, third annular positioning protrusion; 39, second cover plate; 391, third annular positioning groove; 392, second threaded hole. DETAILED DESCRIPTION

[0042] In order to make the technical problems solved by the present application, the technical solutions and the beneficial effects more clearly understood, the present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not used to limit the present application.

[0043] It should be understood that the terms "upper", "lower", "left", "right", "front", "rear", "middle", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations of the present invention.

[0044] like Figure 1 and Figure 2 As shown, the first embodiment of the present invention provides a cell electroporation device, including a first electrode plate 1, a second electrode plate 2, and an electroporation chamber 3. The electroporation chamber 3 is provided with an electroporation chamber 31, an annular channel 32, a flow guiding chamber 33, an inlet 34 connecting the electroporation chamber 31, and an outlet 35 connecting the flow guiding chamber 33. The opposite ends of the annular channel 32 are respectively connected to the electroporation chamber 31 and the flow guiding chamber 33. Preferably, the electroporation chamber 31 is a cylindrical hole. It can be understood that the inlet 34, the electroporation chamber 31, the annular channel 32, the flow guiding chamber 33, and the outlet 35 are sequentially connected.

[0045] The first electrode plate 1 and the second electrode plate 2 are respectively mounted on the two opposite inner sidewalls of the electro-rotation cavity 31; it can be understood that the first electrode plate 1 and the second electrode plate 2 are respectively attached to the upper and lower inner walls of the electro-rotation cavity 31; the first electrode plate 1 and the second electrode plate 2 are arranged in parallel, and one of the first electrode plate 1 and the second electrode plate 2 is a positive electrode plate and the other is a negative electrode plate.

[0046] The axis of the electro-rotating cavity 31, the axis of the annular channel 32, and the center line of the inlet hole 34 coincide.

[0047] In this invention, the electroporation chamber 3 is provided with an inlet 34, an electroporation chamber 31, an annular channel 32, a guide cavity 33, and an outlet 35 connected in sequence. The axis of the electroporation chamber 31 coincides with the center line of the inlet 34. Cell fluid flows into the electroporation chamber 31 through the inlet 34. The electroporation chamber 31 is a cylindrical or frustum-shaped orifice. Figure 7 and Figure 8As shown, the cell solution flows vertically from the center of the electrotransfection cavity 31, and then evenly spreads in all directions (360 degrees) in the radial direction of the electrotransfection cavity 31, and the flow speed in each radial direction is the same. Specifically, the cell solution flows into the electrotransfection cavity 31 from the narrow inlet hole 34, and the flow speed of the cell solution in the inlet hole 34 is large; the space of the electrotransfection cavity 31 is larger, and the flow speed of the cell solution at the inlet of the electrotransfection cavity 31 suddenly decreases, and the flow direction is turned by 90 degrees and evenly spreads, and the flow speed of the cell solution at different radial positions is almost unchanged when the cell solution flows away from the inlet; at the same time, before the cell solution flows into the electrotransfection cavity 31 from the inlet hole 34, the flow direction of the cell solution is along the axial direction of the electrotransfection cavity 31, and with the flow of the cell solution in the electrotransfection cavity 31, the flow direction of the cell solution changes to the radial direction of the electrotransfection cavity 31, and the flow speed of the continuous flow at different horizontal positions is the same; since the flow cytometry electrotransfection device adopts an axisymmetric design, the axis of the electrotransfection cavity 31 is taken as the central axis, and a plurality of coaxial cylindrical surfaces are taken, and through software simulation analysis, the flow speed distribution and size of the cell solution on each cylindrical surface remain the same.

[0048] In addition, as shown in Figure 7 The first electrode sheet 1 and the second electrode sheet 2 are respectively installed on the opposite two inner side walls of the electrotransfection cavity 31; the first electrode sheet 1 and the second electrode sheet 2 are arranged in parallel, and can form a uniformly distributed electric field in the electrotransfection cavity 31, and the electric field line direction is the axial direction of the electrotransfection cavity 31, and the electric field line direction is perpendicular to the flow direction of the cell solution in the electrotransfection cavity 31, so that the electric field intensity of the cells at different positions in the electrotransfection cavity 31 at the same time is consistent, and the uniformity and stability of the cell transfection in the electrotransfection cavity 31 are improved.

[0049] In the present application, the cell solution flows in the radial direction in the electrotransfection cavity 31, and due to the axisymmetric design, the flow of the cell solution in each direction on the same cylindrical surface is equal, and the electric field perpendicular to the flow direction of the cell solution can be formed in the electrotransfection cavity 31, so that the cell solution can be stably and uniformly transfection in the electrotransfection cavity 31; after the cell solution is transfection in the electrotransfection cavity 31, it enters the flow guide cavity 33 from the annular channel 32, and since the axis of the flow guide cavity 33 coincides with the axis of the electrotransfection cavity 31, the flow of the cell solution in each direction of the annular channel 32 is equal; the cell solution is gathered in the flow guide cavity 33 and then discharged through the outlet hole 35.

[0050] In the present application, the cell electrotransfection device has the ability to continuously process large volume of cell liquid, supports long time high-throughput electrotransfection of cells, and has no risk of channel blockage. At the same time, the highly uniform electric field distribution in the electrotransformation cavity 31 and the symmetrical flow field design can ensure that each cell in the continuous flow experiences the same electric field intensity. The continuous flow of cell liquid in the electrotransformation cavity 31 has the same speed size and consistent flow direction in the radial and different height dimensions. The flow direction of the uniform flow is perpendicular to the direction of the uniform electric field, so that each cell in the continuous flow experiences the same number of times and time of electric field shock, ensuring the uniformity of the cell electrotransfection result, thereby improving the overall transfection efficiency and viability.

[0051] In an embodiment, as shown in Figure 5 The annular channel 32 includes a plurality of arc-shaped channels 321, which are annularly spaced around the axis of the electrotransformation cavity 31, and each arc-shaped channel 321 has equal central angle and width. It can be understood that the number of arc-shaped channels 321 can be set according to actual needs, for example, the arc-shaped channels 321 are provided with 4, 6, 8, etc. In the present embodiment, the width and central angle of the plurality of arc-shaped channels 321 are equal, and the centers of the plurality of arc-shaped channels 321 coincide. When the cell liquid in the electrotransformation cavity 31 flows into the flow guide cavity 33 through each arc-shaped channel 321, the flux and speed of the cell liquid in each flow guide cavity 33 are equal, thereby ensuring the stability of the cell liquid aggregation in the flow guide cavity 33 and improving the survival rate of the cells.

[0052] In an embodiment, as shown in Figure 2 and Figure 6 The flow guide cavity 33 is a circular truncated cone cavity, the center line of the inlet hole 34, the axis of the electrotransformation cavity 31, the axis of the circular truncated cone cavity, and the center line of the outlet hole 35 coincide; from one end of the outlet hole 35 towards the annular channel 32, the inner diameter of the circular truncated cone cavity gradually increases. It can be understood that the maximum inner diameter of the circular truncated cone cavity is equal to the inner diameter of the annular channel 32, and the minimum inner diameter of the circular truncated cone cavity is equal to the inner diameter of the outlet hole 35. In the present embodiment, the inner side wall of the circular truncated cone cavity is connected with the inner wall of the annular channel 32. During the process of cell liquid flowing into the circular truncated cone cavity through the annular channel 32, the circular truncated cone cavity can guide the cell liquid to the outlet hole 35, thereby ensuring the stability of the cell liquid discharge from the outlet hole 35.

[0053] In an embodiment, as shown in Figure 3 and Figure 5As shown, the first electrode sheet 1 comprises a first electrode disc 11 and a first electrode lead-out sheet 12 connecting the first electrode disc 11, and the first electrode disc 11 is attached to the top inner wall of the electrotransfection cavity 31; it can be understood that the end of the first electrode lead-out sheet 12 away from the first electrode disc 11 is located outside the electrotransfection cavity 3.

[0054] The second electrode sheet 2 comprises a second electrode disc 21 and a second electrode lead-out sheet 22 connecting the second electrode disc 21, and the second electrode disc 21 is attached to the bottom inner wall of the electrotransfection cavity 31; it can be understood that the end of the second electrode lead-out sheet 22 away from the second electrode disc 21 is located outside the electrotransfection cavity 3.

[0055] The first electrode disc 11 and the second electrode disc 21 are arranged in parallel, the diameter of the first electrode disc 11 is equal to the diameter of the second electrode disc 21, and the axis of the first electrode disc 11, the axis of the second electrode disc 21 and the axis of the electrotransfection cavity 31 coincide. In this embodiment, the first electrode disc 11 and the second electrode disc 21 are coaxially arranged and attached to the upper and lower inner walls of the electrotransfection cavity 31 respectively, so that after the first electrode disc 11 and the second electrode disc 21 are electrified, a uniformly distributed electric field can be formed in the electrotransfection cavity 31, and the direction of the electric field is the axial direction of the electrotransfection cavity 31, which is perpendicular to the flow direction of the cell solution in the electrotransfection cavity 31, thereby ensuring that the electric field intensity experienced by the cells at different positions in the electrotransfection cavity 31 is consistent at the same time, and improving the uniformity and stability of cell transfection in the electrotransfection cavity 31.

[0056] In an embodiment, as shown in Figure 1 As shown, the electrotransfection cavity 3 comprises a first cover plate 36, an electrotransfection block 37, an intermediate block 38 and a second cover plate 39 which are sequentially stacked and installed; the inlet hole 34 is arranged on the first cover plate 36, the electrotransfection cavity 31 is arranged on the electrotransfection block 37, the annular channel 32 is arranged on the intermediate block 38, and the flow guide cavity 33 and the outlet hole 35 are both arranged on the second cover plate 39. It can be understood that the first cover plate 36 and the electrotransfection block 37, the electrotransfection block 37 and the intermediate block 38, and the intermediate block 38 and the second cover plate 39 can be fixed and sealed by ultraviolet curing (UV curing), and can also be fixed and sealed by heat pressing bonding, laser bonding, ultrasonic bonding, hydrophilic treatment bonding (plasma treatment, etc.). In this embodiment, the electrotransfection cavity 3 is designed in a split type by the first cover plate 36, the electrotransfection block 37, the intermediate block 38 and the second cover plate 39, which reduces the manufacturing difficulty and cost of the cell electrotransfection device.

[0057] In an embodiment, as shown in Figure 3 The first cover plate 36 is further provided with a first accommodating groove 361 communicating with the access hole 34, and the first electrode sheet 1 is installed in the first accommodating groove 361. In this embodiment, the first electrode sheet 1 is embedded in the first accommodating groove 361, thereby ensuring the stability of the installation of the first electrode sheet 1 on the first cover plate 36.

[0058] In an embodiment, as shown in Figure 5 The intermediate block 38 is further provided with a second accommodating groove 381, and the annular channel 32 is arranged around the second accommodating groove 381. The second electrode sheet 2 is installed in the second accommodating groove 381. The second electrode sheet 2 is embedded in the second accommodating groove 381, thereby ensuring the stability of the installation of the second electrode sheet 2 on the intermediate block 38.

[0059] In an embodiment, as shown in Figure 2 to Figure 4 The first cover plate 36 is further provided with a first annular positioning groove 362 arranged around the first electrode sheet 1, and the electric rotating block 37 is further provided with a first annular positioning protrusion 371 arranged around the electric rotating cavity 31. The first annular positioning protrusion 371 is inserted into the first annular positioning groove 362. In this embodiment, the first annular positioning protrusion 371 is matched with the first annular positioning groove 362. During the installation of the first cover plate 36 on the electric rotating block 37, the first annular positioning protrusion 371 is inserted into the first annular positioning groove 362, thereby realizing the accurate point position between the first cover plate 36 and the electric rotating block 37.

[0060] In an embodiment, as shown in Figure 2 and Figure 5 The electric rotating block 37 is provided with a second annular positioning groove 372 at an end away from the first annular positioning protrusion 371, and the second annular positioning groove 372 is arranged around the electric rotating cavity 31. The intermediate block 38 is provided with a second annular positioning protrusion 382 arranged around the annular channel 32. The second annular positioning protrusion 382 is inserted into the second annular positioning groove 372. In this embodiment, the second annular positioning protrusion 382 is matched with the second annular positioning groove 372. During the installation of the electric rotating block 37 on the intermediate block 38, the second annular positioning protrusion 382 is inserted into the second annular positioning groove 372, thereby realizing the accurate point position between the electric rotating block 37 and the intermediate block 38.

[0061] In an embodiment, as shown in Figure 2 and Figure 6As shown, the intermediate block 38 is provided with a third annular positioning protrusion 383 at one end away from the second annular positioning protrusion 382, the third annular positioning protrusion 383 is arranged around the annular channel 32, and the second cover plate 39 is further provided with a third annular positioning groove 391 arranged around the flow guide cavity 33; the third annular positioning protrusion 383 is inserted into the third annular positioning groove 391. In this embodiment, the third annular positioning protrusion 383 is matched with the third annular positioning groove 391, and in the process of mounting the intermediate block 38 on the second cover plate 39, the third annular positioning protrusion 383 is inserted into the third annular positioning groove 391, so as to realize the accurate point position between the intermediate block 38 and the second cover plate 39.

[0062] In an embodiment, as shown in Figure 1 As shown, the first cover plate 36 is further provided with a first threaded hole 363, the first threaded hole 363 is communicated with one end of the inlet hole 34 away from the electro-transformation cavity 31; it can be understood that the first threaded hole 363 is arranged at the upper end of the inlet hole 34, and the design of the first threaded hole 363 facilitates the connection of the inlet hole 34 with external pipelines.

[0063] In an embodiment, as shown in Figure 2 As shown, the second cover plate 39 is further provided with a second threaded hole 392, the second threaded hole 392 is communicated with one end of the outlet hole 35 away from the flow guide cavity 33. It can be understood that the second threaded hole 392 is arranged at the lower end of the outlet hole 35, and the design of the second threaded hole 392 facilitates the connection of the outlet hole 35 with external pipelines.

[0064] In an embodiment, as shown in Figure 2 As shown, the first electrode sheet 1 is further provided with a through hole 13, and the inlet hole 34 is communicated with the electro-transformation cavity 31 through the through hole 13. It can be understood that the through hole 13 is arranged at the center position of the first electrode disc 11, and the inner diameter of the through hole 13 is equal to the inner diameter of the inlet hole 34. It is further illustrated that the second electrode sheet 2 includes a completed electrode disc (i.e., a second electrode disc).

[0065] In addition, Figure 9 As shown is the flow velocity and flow direction distribution simulation diagram of the horizontal cross section of the electro-transformation cavity. As shown in Figure 9 As shown in A, the fluid in the central region of the horizontal cross section of the circular electro-transformation cavity 31 just enters from the inlet hole 32, and the flow velocity is large. As the fluid diffuses into the electro-transformation cavity 31, the flow velocity decreases rapidly and gradually remains unchanged. At the same time, as shown in Figure 9As shown in FIG. B, since the fluid enters from the center of the circular electroporation chamber 31, the fluid flows uniformly in all directions (360°) after entering the electroporation chamber 31, that is, the fluid flow rate is the same in any direction in the horizontal plane. Three cylindrical surfaces are taken with different radii with the center of the cylindrical electroporation chamber 31 as the origin, as shown in FIGS. Figure 9 As shown in FIGS. A-C, the simulation results show that the fluid flow rate distribution and size on the three cylindrical surfaces at different positions are consistent. In summary, the high-throughput continuous cell flow in the electroporation chamber 31 has the same flow rate in all directions in the horizontal and vertical dimensions. In addition, the simulation diagram of the electric field distribution and the electric field direction of the vertical section in the center of the electroporation chamber 31 is shown in FIGS. Figure 10 and Figure 11 As shown in FIGS. and, the electric field distribution in the electric conversion region is highly uniform, and the electric field direction is vertically downward, which is perpendicular to the horizontal direction of the continuous fluid in the electric conversion region, ensuring that the electric field intensity experienced by the cells at different positions in the electroporation chamber 31 is consistent at the same time, improving the uniformity and stability of the cell transfection efficiency in the electroporation chamber 31.

[0066] After the cell flow electroporation is completed, the cells flow from the edge of the electroporation chamber 31 through the annular channel 32 of the intermediate layer to the flow guide chamber 33 of the second cover plate 39. Since the four support bars of the intermediate layer are perpendicular to each other, the four arc-shaped channels 321 formed have the same shape and cross-sectional area, and each arc-shaped channel 321 has the same cell flow throughput at the same time. After the four cell flows enter the flow guide chamber 33, they are recombined and discharged from the outlet hole 35.

[0067] The present application proposes a flow cytometry electroporation device that supports long-time high-throughput cell electroporation and reduces the risk of fluid blockage. Secondly, the cell flow injection direction is perpendicular to the fluid flow direction during the electroporation process, and the vertical fluid during the electroporation process is more uniform. At the same time, after the fluid enters the electroporation chamber, it flows in a radial divergent direction (360°), so that the continuous flow flow rate in the horizontal direction during the electric conversion process is the same. In addition, the first electrode sheet 1 and the second electrode sheet 2 are placed in parallel to form a large-volume electroporation chamber 31, and the electric field intensity inside the chamber presents uniform distribution. In summary, thanks to the unique cavity structure design, the present application can ensure the uniformity of the continuous flow in the horizontal and vertical directions during the high-throughput continuous flow electroporation process, and generate a uniform electric field perpendicular to the continuous flow direction in the large-volume electroporation chamber space, so that each cell in the continuous flow experiences the same number of electric shocks and time, ensuring the uniformity of the cell electroporation result, and thereby improving the overall cell transfection rate and viability.

[0068] The beneficial effects of the present application include the following:

[0069] 1. The overall axisymmetric structure design ensures that the fluid continuously enters the electric rotation cavity 31 from the inlet hole, and then flows radially and radially in the horizontal direction 360° in the electric rotation cavity 31, thereby ensuring that the flow rate and flow direction in each direction are the same, and the flow rate of the same horizontal direction at different vertical positions is the same;

[0070] 2. The fluid direction of the continuous flow into the electric rotation cavity 31 is perpendicular to the fluid direction of the electric rotation area, vertically into horizontally, the flow direction changes by 90°, and the vertical component of the flow direction is small;

[0071] 3. Two electrode sheets (i.e. the first electrode sheet 1 and the second electrode sheet 2) are arranged in parallel in the electric rotation cavity 31, and the electric field strength formed after applying voltage is uniformly distributed in the entire electric rotation area, more specifically, the electric field is distributed along the vertical fluid motion direction, and is perpendicular to the horizontal direction of the continuous flow of the electric rotation area;

[0072] 4. The present application ensures that each cell in the continuous flow experiences the same number of electric shocks and time when flowing through the electric rotation area, which includes: uniform electric field distribution in the vertical direction in the electric rotation area, and uniform flow rate distribution in the horizontal direction along different radial angles; The overall ensures the uniformity and stability of the performance of the large volume cells in the flow electroporation, and improves the overall transfection efficiency and viability of the cell flow.

[0073] The above is only an embodiment of the cell electroporation device of the present application, and does not limit the present application. Any modification, equivalent replacement and improvement within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A flow cytometric electroporation device, characterized by, The application relates to an electric conversion cavity body, which comprises a first electrode sheet, a second electrode sheet and an electric conversion cavity; an electric conversion cavity, an annular channel, a flow guide cavity, an inlet hole communicating with the electric conversion cavity and an outlet hole communicating with the flow guide cavity are arranged in the electric conversion cavity; opposite ends of the annular channel are respectively communicated with the electric conversion cavity and the flow guide cavity; The first electrode sheet and the second electrode sheet are respectively arranged on opposite inner walls of the electric conversion cavity; The axis of the electric conversion cavity, the axis of the annular channel and the center line of the inlet hole are coincident.

2. The flow cytometric electrotransfection device of claim 1, wherein, The annular channel comprises a plurality of arc-shaped channels, the arc-shaped channels are annularly and spacedly arranged around the axis of the electric conversion cavity, and the central angle and the width of each arc-shaped channel are equal.

3. The flow cytometric electrotransfection device of claim 1, wherein, The flow guide cavity is a circular truncated cone cavity, the center line of the outlet hole, the axis of the electric conversion cavity, the axis of the circular truncated cone cavity and the center line of the outlet hole are coincident; the inner diameter of the circular truncated cone cavity gradually increases from the outlet hole to one end of the annular channel.

4. The flow cytometric electrotransfection apparatus of claim 1, wherein, The first electrode sheet comprises a first electrode disc and a first electrode lead-out sheet connected with the first electrode disc, and the first electrode disc is attached to the top inner wall of the electric conversion cavity; The second electrode sheet comprises a second electrode disc and a second electrode lead-out sheet connected with the second electrode disc, and the second electrode disc is attached to the bottom inner wall of the electric conversion cavity; The first electrode disc and the second electrode disc are arranged in parallel, the diameter of the first electrode disc is equal to the diameter of the second electrode disc, and the axis of the first electrode disc, the axis of the second electrode disc and the axis of the electric conversion cavity are coincident.

5. The flow cytometric electrotransfection device of claim 1, wherein, The electric conversion cavity body comprises a first cover plate, an electric conversion block, an intermediate block and a second cover plate which are sequentially and laminatedly arranged; the inlet hole is arranged on the first cover plate, the electric conversion cavity is arranged on the electric conversion block, the annular channel is arranged on the intermediate block, and the flow guide cavity and the outlet hole are arranged on the second cover plate.

6. The flow cytometric electrotransfection device of claim 5, wherein, The first cover plate is further provided with a first accommodating groove communicated with the inlet hole, and the first electrode sheet is arranged in the first accommodating groove; The intermediate block is further provided with a second accommodating groove, the annular channel is arranged around the second accommodating groove, and the second electrode sheet is arranged in the second accommodating groove.

7. The flow cytometric electrotransfection device of claim 5, wherein, The first cover plate is further provided with a first annular positioning groove arranged around the first electrode sheet, and the electric conversion block is further provided with a first annular positioning convex arranged around the electric conversion cavity; the first annular positioning convex is inserted into the first annular positioning groove; The end of the electric conversion block away from the first annular positioning convex is provided with a second annular positioning groove arranged around the electric conversion cavity, and the intermediate block is provided with a second annular positioning convex arranged around the annular channel; the second annular positioning convex is inserted into the second annular positioning groove; The end of the intermediate block away from the second annular positioning convex is provided with a third annular positioning convex arranged around the annular channel, and the second cover plate is further provided with a third annular positioning groove arranged around the flow guide cavity; the third annular positioning convex is inserted into the third annular positioning groove.

8. The flow cytometric electrotransfection device of claim 5, wherein, The first cover plate is further provided with a first threaded hole, which is communicated with one end of the inlet hole away from the electric rotation cavity; and / or The second cover plate is further provided with a second threaded hole, which is communicated with one end of the outlet hole away from the flow guide cavity.

9. The flow cytometric electrotransfection apparatus of any one of claims 1 to 8, wherein, The first electrode sheet is further provided with a through hole, and the inlet hole is communicated with the electric rotation cavity through the through hole.

10. The flow cytometric electrotransfection apparatus of any one of claims 1 to 8, wherein, The electric rotation cavity is in a columnar shape.

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