KR20200041760A - Pneumatically-driven Cell Concentrator, Cell Concentrate Method, Method of Manufacturing Microfluidic Channel and Method of Manufacturing Pneumatic Valves - Google Patents

Abstract

The present invention relates to a pneumatically-driven cell concentrator, a cell concentration method using same, a method for producing a microfluidic channel, and a method for producing a pneumatic valve. The pneumatically-driven cell concentrator comprises: an inlet port to which a fluid containing cells is injected; a microfluidic channel through which the fluid injected through the inlet port flows; a plurality of pneumatic valves which controls the concentration of cells flowing through the microfluidic channel, and the flow of the cells or the fluid; a pneumatic port which is connected to each of the pneumatic valves to drive the pneumatic valves; and an outlet port which includes a fluid outlet port (Of) for discharging only the fluid so as to separately discharge the concentrated cells and the fluid while passing through the pneumatic valves, and a cell outlet port (Oc) for discharging the fluid including the concentrated cells. Accordingly, due to a high processing speed, the pneumatically-driven cell concentrator can be widely applied to industries requiring large-scale cell concentration.

Description

Pneumatically-driven Cell Concentrator, Cell Concentrate Method, Method of Manufacturing Microfluidic Channel and Method of Manufacturing Pneumatic Valves

The present invention relates to a pneumatically driven cell concentrator, and more specifically, by collecting and concentrating a large number of cells using a pneumatically driven system, the processing speed is fast, and the pneumatically driven system can be applied to industries requiring large-scale cell concentration. It relates to a cell concentrator, a cell concentration method using the same, a method for manufacturing a microfluidic channel, and a method for manufacturing a pneumatic valve.

With the development of microfluidic engineering and BioMEMS technology, devices for trapping cells for biological and cellular research have been developed using a variety of microfluidic platforms.

The method of capturing the cells is mainly a method of capturing a single cell using a cup-like structure, a double groove structure, a microchamber array, a micro-obstruction, ultrasound, an optical tweezer, dielectrophoresis, and magnetic force. Since most of these capture methods have a disadvantage of slow processing speed, they are limited to be used for the concentration of harmful algae such as ballast water that requires the concentration of large-capacity cells.

Recently, a study on the capture of a pneumatically driven method for single cell capture using a simple fluid flow has been reported. However, this is only a single cell capture technique, and there is no research to capture and concentrate a large number of target cells and cells, so there is a limit to industrial application.

Accordingly, the present applicant has developed an active concentrating device of a pneumatically driven method for concentrating a large number of cells, and has completed this to propose the present invention.

[Non-Patent Document 1] D. D. Carlo, N. Aghdam, and Luke P. Lee, Anal. Chem., Vol. 78, pp. 4925-4930, 2006. [Non-Patent Document 2] M. Khabiry, B. G. Chung, M. J. Hancock, H. C. Soundararajan, Y. Du, D. Cropek, W. G. Lee, and A. Khademhosseini, Small, Vol. 5, No. 10, pp. 1186-1194, 2009. [Non-Patent Document 3] S. Yamamura, H. Kishi, Y. Tokimitsu, S. Kondo, R. Honda, SR Rao, M. Omori, E. Tamiya, and A. Muraguchi, Single-Cell Microarray for Analyzing Cellular Response , Anal. Chem., Vol. 77, pp. 8050-8056, 2005. [Non-Patent Document 4] L. R. Huang, E. C. Cox, R. H. Austin, J. C. Sturm, Continuous particle separation through deterministic lateral displacement, Science. Vol. 304 (5673), pp. 987-90, 2004. [Non-Patent Document 5] F. Petersson, A. Nilsson, C. Holm, H. J *? * Nsson and T. Laurell, Continuous separation of lipid particles from erythrocytes by means of laminar flow and acoustic standing wave forces. Lab Chip, Vol. 5, 20-22, 2005. [Non-Patent Document 6] P. L. Johansen, F. Fenaroli, L. Evensen, G. Griffiths, and G. Kostera, Optical micromanipulation of nanoparticles and cells inside living zebrafish, Nat Commun. Vol. 7, 10974, 2016.

The present invention has been devised to solve the above problems, a Sieve valve that filters out and concentrates only the cells in the working fluid, a fluid discharge valve that discharges only the working fluid while concentrating the cells, and a cell collection valve for obtaining concentrated cells It is an object of the present invention to provide a pneumatically driven cell concentrator that can be applied to industries requiring fast concentration of large cells by collecting and concentrating a large number of cells using a pneumatically driven method.

In addition, there is an object to provide a cell concentration method using the cell concentrator as described above.

In addition, the object of the present invention is to provide a method for manufacturing a microfluidic channel and a pneumatic valve of a cell concentrator.

In order to achieve the above object, in the present invention, the inlet port through which the fluid containing cells is injected, the microfluidic channel through which the fluid injected through the inlet flows, and the concentration of the cells flowing through the microfluidic channel , A plurality of pneumatic valves for controlling the flow of fluid or cells, and a pneumatic port (Pneumatic port) connected to each pneumatic valve to drive the pneumatic valve, and the concentrated cells and fluid passing through the pneumatic valve Provided is a pneumatically driven cell concentrator that includes a fluid outlet (Of) that discharges only fluid to separate and discharge, and an outlet port that includes a cell outlet (Oc) that discharges fluid containing concentrated cells do.

The pneumatic valve is a device that controls the microfluidic channel, a sieve valve (Vs) for filtering and concentrating cells in the working fluid, and a fluid for controlling to discharge the working fluid (Qf) while the cells are concentrated It may be composed of a control valve (Vf) and a cell collection valve (Vc) that controls to obtain a cell concentrate (Qc).

Here, the filter valve is formed by a valve diaphragm (Valve diaphragm) that can be deformed by receiving external compressed air, a dome-shape fluid chamber, and an interconnection channel formed at a lower end of the hemispherical fluid chamber ).

The microfluidic channel is connected to the inlet, the upper microfluidic channel through which the cell-containing fluid Qfc flows, the channel through which the concentrated cells Qc flow, and the fluid filtered by the cell during the concentration process ( It may include an interconnection channel (Qf) is divided into a flowing fluid channel.

In the present invention, the lower portion of the upper microfluidic channel is characterized in that a built-in channel of a right angle shape through which only fluid can pass is integrally formed.

In addition, the upper microfluidic channel may be formed in a streamlined shape in which at least two curved portions are formed so that cells are concentrated while fluid is discharged through the internal channel.

On the other hand, the cell concentrator of the present invention has a multi-layer structure, and the inlet, outlet, and pneumatic connectors are formed to form a fluid and pneumatic supply layer for external fluid connection and pneumatic connection, and a valve membrane of the pneumatic valve Valve diaphragm layer for, a 3D microfluidic channel network layer in which the microfluidic channel is formed (3-dimensional channel network layer), and additionally provided below the 3D microfluidic channel network layer And an additional sealing layer.

In the present invention, the 3D microfluidic channel network layer is an upper layer on which a microfluidic channel for injecting and discharging fluid containing cells is formed, and an interconnection for forming an interconnection structure between the pneumatic valves. It may be formed of a lower layer in which a channel is formed.

On the other hand, in order to achieve the object of the present invention as described above, the step of introducing a fluid containing cells through an inlet port (inlet port), and the valve membrane (Valve diaphragm) is provided with external compressed air to the filter valve (Compressed air) When) is applied, the valve membrane (Valve diaphragm) is deformed, and the outlet surface of the fluid channel formed on the inclined surface connecting the microfluidic channel and the hemispherical fluid chamber is narrowed, blocking the cells, and allowing the fluid to be discharged ( Sieve surface) is generated, the fluid control valve (Vf) that only flows off the fluid, and the cell collection valve (controlling the passage through which the fluid containing the concentrated cells flows to obtain the cell concentrate (Qc) ( By controlling Vc) to ON, respectively, the step of concentrating the cells, the external compressed air applied to the filter valve Vs is released (OFF), and the channel through which the concentrated cells Qc flows are opened and fluidized. To block the channel of (Qf), by controlling the cell collection valve (Vc) OFF, by controlling the fluid control valve (Vf) to ON, the pneumatically driven cell concentrator proceeding, including the step of discharging the concentrated cells to the outside Cell concentration method using is provided.

The present invention is characterized in that by draining the fluid through the embedded channel while the cells are concentrated, the cells are deformed to prevent clogging the channels.

In addition, the cell concentration method of the present invention can control the deformation of the valve membrane and control cells of various sizes by controlling the pressure applied to the manure valve Vs according to the size of the cells.

In addition, in order to achieve the object of the present invention as described above, a first step of applying a liquid resin to a first mold for making a hemispherical fluid chamber to a predetermined thickness, and the first step in which the liquid resin is applied in the first step A second step of curing the mold, a third step of applying a liquid resin to a second mold for forming a right-angled embedded channel with a predetermined thickness, and a second mold in which the liquid resin is applied in the third step Atmospheric plasma in the fourth curing step and the first curing resin in which the air cavity is formed by removing the first mold from the first curing resin cured in the second step, and the second curing resin cured in the fourth step A fifth step of bonding using, a second step of removing the second mold from the second cured resin and bonding the bonded cured resin to a glass substrate using atmospheric pressure plasma, and a three-dimensional microfluidic channel Yeah A seventh step of aligning the cured resin after applying a liquid resin to a third mold for manufacturing a lower layer of a 3-dimensional channel network layer, and an eighth step of curing the liquid resin of the seventh step, The ninth step in which the deformed reverse phase is replicated to the liquid resin as the air inside the air cavity, which is a closed space, expands, and the resin film is swollen, and the reverse phase of the deformation of the resin film is cured while being cured to form a right angle. There is provided a method of manufacturing a pneumatically driven cell concentrator microfluidic channel comprising a tenth step in which a microfluidic channel in which an embedded channel is formed is completed.

In addition, in order to achieve the object of the present invention as described above, a first step of applying a liquid resin to a first mold for making a hemispherical fluid chamber with a predetermined thickness, and a first step of applying the liquid resin in the first step Step II for curing the mold, Step III for applying the liquid resin to the second mold with a predetermined thickness, Step IV for curing the second mold coated with the liquid resin in the Step III, and Step II A fifth step of bonding the first cured resin cured in the step to the second cured resin cured in the step using atmospheric pressure plasma, and then bonding the glass substrate to the glass substrate using atmospheric pressure plasma. Step VI to cure the liquid resin of the air, and the air inside the air cavity (air cavity), which is a closed space, expands, and as the resin film swells, the deformed reversed phase is replicated to the liquid resin and the lower fine A pneumatically driven cell concentrator pneumatic comprising a decontamination step in contact with a mold structure for fabricating a fluid channel network layer to form a through structure, and a decontamination step in which the through structure is cured to complete a pneumatic valve structure. A valve manufacturing method is provided.

According to the present invention as described above, a sieve valve that filters and concentrates only cells in the working fluid, a fluid discharge valve that discharges only the working fluid while concentrating the cells, and a cell collecting valve for obtaining concentrated cells include a pneumatic driving method By collecting and concentrating a large number of cells, the processing speed is fast, and thus, it can be applied to industries that require large-scale cell concentration.

1 is an overall perspective view showing a cell concentrator of the pneumatic drive method of the present invention.
Figure 2 is an exploded perspective view showing a cell concentrator of the pneumatic drive method of the present invention.
3 is a plan view showing a fluid and pneumatic supply layer in the present invention.
4 is a plan view showing a three-dimensional microfluidic channel network layer in the present invention.
5 is a plan view of the network layer of FIG. 4 divided into an upper layer and a lower layer.
Figure 6 is a perspective view showing the structure of the manure valve for cell concentration of the present invention.
7 is a perspective view showing the concentration of cells introduced into the filter of the filter for cell concentration of the present invention.
Figure 8 is a perspective view showing the discharge of the concentrated cells in the manure valve for cell concentration of the present invention.
9 is a process diagram showing a method of manufacturing a microfluidic channel of the present invention.
10 is a process diagram showing a method of manufacturing a three-dimensional microfluidic channel network layer (3-dimensional channel network layer) pneumatic valve of the present invention.
11 is a photomicrograph of a concentrator for cell concentration of the present invention.
FIG. 12 is an SEM photograph showing an AA cross section of a particle collection channel in the photograph of FIG. 11.
FIG. 13 is an SEM photograph showing a BB cross section of a hemispherical filter valve (Sieve Valve) for capturing cells in the photograph of FIG. 11.
14 is a graph showing the results of flow measurement measured at two outlet ports according to the pneumatic valve operation of the present invention.
Figure 15 is a cell concentration principle verification test of the present invention, (a) the initial state, (b) the concentration process, (c) is a photograph showing after discharge.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the present embodiments allow the disclosure of the present invention to be complete, and the scope of the invention to those skilled in the art is completely It is provided to inform you.

1 is an overall perspective view showing a cell concentrator of a pneumatic drive method of the present invention, FIG. 2 is an exploded perspective view showing a cell concentrator of a pneumatic drive method of the present invention, and FIG. 3 shows a fluid and pneumatic supply layer in the present invention 4 is a plan view showing a three-dimensional microfluidic channel network layer in the present invention.

As shown in FIG. 1, the pneumatically driven cell concentrator of the present invention is an inlet port 12 through which fluid containing cells is injected, and a microfluidic fluid through which the fluid injected through the inlet 12 flows. Channel 32, a plurality of pneumatic valves (50) (54) (56) for controlling the concentration of cells flowing through the microfluidic channel (32), fluid or cell flow, and the pneumatic valve (50) ( The pneumatic ports 16, 17, 18 connected to the respective pneumatic valves 50, 52, 54 to drive the 52) 54, and concentrated while passing through the pneumatic valve Outlet port including a fluid outlet (Of) 14 for discharging only the fluid to separate the cells and the fluid, and a cell outlet (Oc) 15 for discharging the fluid containing the concentrated cells ( 14) (15).

As shown in Figure 2, the cell concentrator of the present invention is characterized by having a multi-layer structure, and the embodiment in the present invention constitutes a cell concentrator by combining four layers.

The cell concentrator of the present invention is largely a fluid and pneumatic supply layer (10), a valve diaphragm layer (20), and a three-dimensional microfluidic channel network layer (3-dimensional channel network layer) 30 and an additional sealing layer 40 provided under the 3D microfluidic channel network layer 30.

Here, each layer includes the components of the present invention, the fluid and pneumatic supply layer (pneumatic supply layer) 10 is the inlet 12, the outlet 14, 15 and the pneumatic connector (17) ( 18) is formed to allow external fluid connection and pneumatic connection.

The valve diaphragm layer 20 is a layer for forming a valve membrane of the pneumatic valves 50, 54, 56, and a 3-dimensional channel network layer (3-dimensional channel network layer) ( 30) is a layer for forming the microfluidic channel 32.

The pneumatic valve in the present invention is a device that controls the microfluidic channel 32, a sieve valve (Vs) 50 for filtering and concentrating cells in the working fluid, and a working fluid while the cells are concentrated It consists of a fluid control valve (Vf) 56 which controls to discharge (Qf), and a cell collection valve (Vc) 54 that controls to obtain a cell concentrate (Qc).

The three valves control each microfluidic channel (32, 34), a sieve valve (Vs) 50 to filter and concentrate cells in the working fluid, and a working fluid while the cells are concentrated The valve (Vf) 56 that controls to discharge (Qf) and the valve (Vc) 54 that controls to obtain a cell concentrate (Qc) are utilized to concentrate and discharge the introduced cells.

The microfluidic channel 32 is connected to the inlet 12, an upper microfluidic channel 32 through which a fluid Qfc containing cells is introduced, and a channel through which the concentrated cells Qc flow ( 34b) and an interconnection channel 34 formed of a fluid channel 34a through which the cell filtered fluid Qf flows during concentration.

In the present invention, the lower portion of the upper microfluidic channel 32 is integrally formed with an embedded channel 32a through which only fluid can flow, the embedded channel 32a as shown in FIG. 6. , Form a right angle, and cells are concentrated while the fluid is discharged through the built-in channel 32a.

In addition, the upper microfluidic channel 32 is preferably made of a streamlined shape in which at least two bends are formed such that cells are concentrated while fluid is discharged through the embedded channel 32a.

In the present invention, one inlet port 12 and two outlet ports 14 and 15 for injecting and discharging the fluid are constituted, and the fluid outlet (Of) 14 discharges only the fluid. ) And the outlet (Oc) 15 through which the fluid containing the concentrated cells are discharged.

Here, the filter valve 50 includes a valve diaphragm 20 that can be deformed by receiving external compressed air, as shown in FIG. 6, a dome-shape fluid chamber 52 It comprises a hole (52a) for connecting to the interconnection channel (interconnection channel) 34 at the lower end of the.

Concentration principle

Figure 6 is a perspective view showing the structure of the manure valve for cell enrichment of the present invention, showing the structure of the Sieve valve and the fluid channel network of the cell concentrator which is a core element of the present invention. The filter valve (Vs) is composed of a valve membrane (Valve diaphragm) 20 that can be deformed by receiving external compressed air, and a dome-shape fluid chamber (52). In addition, unlike the general pneumatic valve structure manufactured on the same plane, in the present invention, the upper microfluidic channel (32) into which the cell-containing fluid (Qfc) flows, and the channel from the upper side by cell deformation An embedded channel 32a through which only fluid can flow to prevent clogging of the channel, and two interconnection channels formed at the bottom, that is, a channel 34b through which concentrated cells Qc flow and concentration The process consists of a channel (34a) through which the cell filtered fluid (Qf) flows. Three fluid channels located in the upper and lower parts are simultaneously manufactured in the process of manufacturing a dome-shape fluid chamber to increase the efficiency of the process.

Figure 7 is a perspective view showing the concentration of cells introduced to the manure valve for cell concentration of the present invention, the flow of fluid using the fluid valve (Vf), cell valve (Vc) and Sieve valve (Vs) during cell concentration (Qfc) Represents the process of concentrating cells in and discharging only the working fluid.

In the present invention, when the fluid mixture containing cells is introduced into the cell concentrator through the inlet port 12, external compressed air is compressed in the filter valve 50 equipped with a valve diaphragm. air) to drive the Sieve valve, turn off the fluid control valve (Vf) that only flows fluid, and collect cells that control the passage through which the fluid containing the concentrated cells flows to obtain the cell concentrate (Qc). The cells are concentrated by controlling the valves Vc to ON, respectively.

At this time, when external compressed air is applied to the filter valve equipped with a valve diaphragm, the valve membrane (Valve diaphragm) 20 is deformed, as shown in FIG. 7, to form a microfluidic channel and a hemispherical fluid. The outlet surface of the fluid channel formed on the inclined surface to which the chamber is connected becomes narrow. That is, the cells are blocked, but a surface (Sieve surface) through which the fluid can be discharged is generated, so that only the cells are concentrated. At this time, since there is continuous fluid discharge through an embedded channel 32a through which only fluid can flow, cells are deformed to prevent clogging of the channel. In addition, by controlling the pressure applied to Vs according to the size of the cells, it is possible to control the deformation of the valve membrane and to concentrate cells of various sizes.

Since the curved surface of the hemispherical fluid chamber 52 is replicated from the deformed form of the valve membrane 20, it is possible to efficiently control the deformation of the valve membrane 20 at low pressure. The valve for the cell concentration, while driving the Vs, at the same time blocking the channel through which the concentrated cells (Qc) flow and blocking the channel through which only the fluid (Qf) in the enrichment process is blocked, and the fluid in which the cells are caught in the enrichment process ( Cell control is performed by controlling Vp and Vf, respectively, to open channels that only flow Qf).

Figure 8 is a perspective view showing the discharge of the concentrated cells in the manure valve for cell concentration of the present invention, the discharge process of the concentrated cells. Cell capture valve (Vc) to release the external compressed air (OFF) that was applied to the filter valve (Vs) for cell concentration, and at the same time open the channel through which the concentrated cells (Qc) flow and block the channel of the fluid (Qf). ) To OFF, and the fluid control valve (Vf) to ON to discharge the concentrated cells to the outside.

FIG. 5 is a plan view of the network layer of FIG. 4 divided into an upper layer and a lower layer, wherein the 3D microfluidic channel network layer 30 is formed with an upper microfluidic channel 32 for injecting and discharging fluid containing cells. It can be made of an upper layer (30a) and the lower layer (30b) is formed with interconnection channels (34a) (34b) for forming an interconnection (interconnection) structure between the pneumatic valve.

The three-dimensional microfluidic channel network layer 30 utilizes two SU-8 mold structures and connects three-dimensionally to inject fluid containing cells and discharge fluid and cells, respectively, by the process proposed in the present invention. Build the structure.

9 is a process chart showing a method of manufacturing a microfluidic channel of the present invention, and FIG. 10 is a process chart showing a method of manufacturing a 3D microfluidic channel network layer (3-dimensional channel network layer) pneumatic valve of the present invention.

First, a method of manufacturing a microfluidic channel includes a first step of applying a liquid resin 112 to a first mold 110 for making a hemispherical fluid chamber with a predetermined thickness, and the liquid resin 112 in the first step. A second step of curing the applied first mold, a third step of applying a liquid resin to a second mold for forming a right-angled embedded channel with a predetermined thickness, and the liquid resin 114 in the third step The first step of curing the second mold is applied, and the first curing resin (air cavity) (115) is formed by removing the first mold from the first curing resin (112a) cured in the second step ( A fifth step of bonding 112a) to the second cured resin 114a cured in the fourth step using atmospheric pressure plasma, and removing the second mold from the second cured resin 114a and bonding the cured resin 118 The 6th step of bonding to the glass substrate (Glass substrate) using atmospheric pressure plasma, and the third The seventh step and the seventh step of aligning the cured resin 118 after applying the liquid resin 117 to the third mold for fabricating the lower layer of the 3-dimensional channel network layer The eighth step of curing the resin, and the air inside the air cavity (air cavity) 115, which is a closed space, expands, and the resin film swells, resulting in the deformed reverse phase being replicated to the liquid resin 117. , It is composed of a tenth step in which the microfluidic channel 117a in which a right-angled internal channel is formed by curing and curing the reverse phase of the deformation of the resin film is completed.

Specifically, in the present invention, the microfluidic channel is (1) 80 µm of liquid PDMS is applied to the SU-8 mold for making a domed fluid chamber, and (2) 90 of the SU-8 mold coated with PDMS in (1) is applied. Cure for 30 minutes at ℃, (3) Apply liquid PDMS to SU-8 different from (1) to a thickness of 80 µm, and (4) Apply SUMS mold with PDMS in (3) process at 90 ℃ for 30 minutes Curing, (5) bonding the PDMS cured in the process (2) using normal pressure plasma to the PDMS cured in the process (4), and (6) bonding the bonded PDMS horizontally and vertically to the glass substrate of 18 mm each. Plasma bonding using plasma, (7) 0.3 mL of liquid PDMS is applied to the SU-8 mold for fabrication of the lower 3-dimensional channel network layer, and then the PDMS mold in the process (6) is aligned. , (8) (7) curing process at 130 ℃ for 30 minutes, (9) the air inside the closed space air cavity expands, and the PDMS membrane swells. Replication in the liquid PDMS, (10) shows the shape of the structure production is completed.

In addition, the 3D microfluidic channel channel layer (3-dimensional channel network layer) pneumatic valve is a first step of applying a liquid resin 212 to the first mold 210 for making a hemispherical fluid chamber to a predetermined thickness, and In the first step, the second step of curing the first mold coated with the liquid resin 212, the third step of applying the liquid resin 214 to the second mold with a predetermined thickness, and in the third step, the The normal pressure plasma is applied to the second curing resin 214a cured in the fourth step and the first curing resin 212a cured in the second step to cure the second mold coated with the liquid resin 214. After bonding using a fifth step of bonding using a normal pressure plasma to a glass substrate (Glass substrate), a sixth step of curing the liquid resin of the fifth step, and a closed space air cavity (air cavity) 215 The air inside expands, causing the resin film to swell. As the deformed reversed phase is replicated to the liquid resin, it is brought into contact with the mold structure for fabrication of the lower microfluidic channel network layer, and a step of forming a through structure is formed, and the through structure is cured to form a pneumatic valve 217a. It includes the final step of completion.

Specifically, the manufacture of the three-dimensional microfluidic channel network layer valve of the present invention includes (I) 80 μm of liquid PDMS applied to the SU-8 mold for making a domed fluid chamber, (II) ( Curing the SU-8 mold coated with PDMS in step I) at 90 ° C for 30 minutes, applying liquid PDMS to SU-8 different from (III) (I) to a thickness of 80 μm, (IV) (III) The SU-8 mold coated with PDMS is cured at 90 ° C for 30 minutes, and the PDMS cured in the (V) (II) process is bonded to the PMDS cured in the (IV) process using normal pressure plasma, and then again 18 mm Bonding using atmospheric pressure plasma on the glass substrate, (VI) curing at 130 ℃ for 30 minutes, (VII) the air inside the closed space air cavity expands, causing the PDMS film to swell, and the reverse phase of the deformation of the PDMS film is liquid PDMS Replicating at this time, in contact with the SU-8 mold structure for fabricating the lower microfluidic channel network layer, forming a through structure, (VIII) This is the shape of the structure that has been produced.

The microfluidic channel, embedded channel, hemispherical fluid chamber, and three-dimensional microfluidic channel network layer of the present invention utilize two SU-8 molds to form a hemispherical fluid chamber ( Formation of a half-spherical dome shape fluid chamber structure and a 3-dimensional channel network layer are fabricated.

The remaining three PDMS layers are fabricated as follows. The layer for external fluid and pneumatic connection (Fluid & pneumatic supply layer) was prepared by applying and curing 10 mL of liquid PDMS to the SU-8 mold. And the valve membrane (Valve diaphragm layer) was prepared by applying and curing liquid PDMS to a thickness of 80 μm on a 4 inch silicon wafer. In the case of an additional sealing layer, it was prepared by applying and curing 10 mL of liquid PDMS on a 4 inch silicon wafer. Curing conditions for all processes were cured at 90 ° C for 30 minutes using a hot plate. When all components of FIG. 2 are completed, sequential bonding is performed using atmospheric pressure plasma to complete the platform.

Prototype

FIG. 11 is a micrograph of a concentrator for cell concentration of the present invention, FIG. 12 is an SEM photograph showing an AA cross section of a particle collection channel in the photo of FIG. 11, and FIG. 13 is a cell in the photo of FIG. 11 This is a SEM photograph showing the BB cross-section of a hemispherical filter valve for capture.

12 is a cell collection channel (cell collection channel), a structure that discharges the fluid through the Only fluid channel while concentrating the cells after the Sieve valve operation. Since the inlet port channel and the outlet port channel are simultaneously formed during the production of a dome shape fluid chamber among the Sieve valve structures, they have a curved cross-section, not a right-angle cross-section, and are connected to the top of the fluid chamber (FIG. 13).

In the photo, 1 is a fluid channel connected to the inlet, 2 is a right-angle fluid channel through which only fluid can pass, 3 is a fluid chamber located under the enrichment valve, 4 is an interconnection channel, and 5 is an interconnection channel.

[Table 1] shows the size of the detailed structure using the main component names and SEM.

designation W or D (μm) H (μm) R (μm) One Fluid channel connected to inlet 342 41 496.84 2 Right-angled fluid channel through which only fluid can pass 24 5.35 3 Fluid chamber located under the concentration valve 840 165 979.12 4 Hall for interconnection channel and connection 400 (D) 5 interconnection channel 100 40

(W: Width, D: Diameter, H: Height, R: Radius of curvature)

Concentration test

14 is a graph showing the flow measurement result measured at two outlet ports according to the pneumatic valve operation of the present invention, and when the valve is operated as shown in [Table 2], the flow rate from the two outlet ports of the cell concentrator This is the result of measuring the flow rate using a sensor. By controlling the solenoid valve with the Labview program using a laptop computer, each of the three valves was individually applied with external compressed air. In the basic experiment, when cells were mixed in the fluid, the flow sensor was difficult to operate, so the test was performed using pure water.

For sample loading, in the case of a state, since all valves are off (1 signal), the flow rate is measured at two outlet ports. When operating the Sieve Valve (V1) for cell concentration (1 signal & b state), the flow rate of the Sieve valve chamber is reduced because the fluid passage through the Sieve valve chamber is reduced. In this state, when air pressure is applied to the valve for the cell discharge, that is, Vc (3 signal), the passage for the cell discharge is completely blocked, so all the introduced fluid flows into the outlet port for fluid discharge, so only Qf It is measured, and in the case of Qc, a result close to 0 is obtained (c state). In the opposite case, only Qc can be obtained, and Qf can implement a d state that cannot be measured.

Through this experiment, it can be confirmed that the Sieve valve and the valve for controlling two fluid passages were successfully manufactured.

Table 2 shows the pneumatic valve conditions for the cell concentration test according to the conditions of FIG. 3.

Platform Operation Valve Operation Signal Sieve Fluid Particle state Operation Flowrate Vs Vf Vp a Sample loading Q _f , Q _c 4 OFF OFF OFF b Only sieving Q _f , Q _c One ON OFF OFF c Sieving / Wasting Q _f 2 ON OFF ON d Sieving / Collecting Q _c 3 ON ON OFF

Cell concentration principle verification

15 is a cell concentration principle verification test of the present invention, (a) the initial state, (b) the concentration process, (c) is a photograph showing the discharge.

In order to prove the concentration principle suggested in the present invention, experiments were conducted as shown in [Table 2]. In this experiment, Heterocapsa triquetra with a diameter of about 10 μm was used. After flowing the initial fluid to make the fluid channel existing inside the chip wet, a 1 meter tube was connected to each outlet port. A fluid mixed with cells is injected into the platform using a head difference of 60 cm H2O (a). When the valve for driving the Sieve valve and discharging only the fluid separated from the cell is opened, and the valve for discharging the cell is closed, the cells are concentrated in the enrichment channel (b). After concentration, when the fluid discharge valve is closed and the cell discharge valve is opened, the concentrated cells are discharged (c). The pressure applied to the three valves is 16 kPa. Predicted by pictures, it is estimated that about 4200 cells are concentrated.

Cells could be successfully concentrated using the structure proposed in this study.

The rights of the present invention are not limited to the embodiments described above, but are defined by the claims, and those skilled in the art can make various modifications and adaptations within the scope of the claims. It is self-evident.

10: fluid and pneumatic supply layer
12: Inlet port 14: Fluid outlet (Of)
15: cell outlet (Oc)
16, 17, 18: Pneumatic port
20: Valve diaphragm layer
30: 3-dimensional channel channel layer (3-dimensional channel network layer)
32: microfluidic channel 34: interconnection channel
40: additional sealing layer
50: Sieve Valve (Vs) 50
54: cell collection valve (Vc)
56: fluid control valve (Vf)

Claims (13)

An inlet port into which a fluid containing cells is injected;
A microfluidic channel through which the fluid injected through the inlet flows;
A plurality of pneumatic valves for controlling concentration of cells flowing in the microfluidic channel, fluid or flow of cells;
A pneumatic port connected to each pneumatic valve to drive the pneumatic valve; And
Outlet port including a fluid outlet (Of) for discharging only the fluid and a cell outlet (Oc) for discharging the fluid containing the concentrated cells so as to separate and discharge the concentrated cells and fluid while passing through the pneumatic valve. );
Cell concentrator of the pneumatic drive method comprising a. The method according to claim 1,
The pneumatic valve is an element that controls the microfluidic channel, and sieve valve (Vs) for filtering and concentrating cells in the working fluid;
A fluid control valve (Vf) that controls to discharge the working fluid (Qf) while the cells are concentrated; And
A cell collection valve (Vc) that controls to obtain a cell concentrate (Qc);
Cell concentrator of the pneumatic drive method, characterized in that consisting of. The method according to claim 2,
The filter valve may be deformed by receiving external compressed air (Valve diaphragm);
Dome-shape fluid chamber; And
It is formed at the lower end of the hemispherical fluid chamber, the pneumatically driven cell concentrator, characterized in that it comprises a hole for connecting with an interconnection channel (interconnection channel). The method according to claim 1,
The microfluidic channel is an upper microfluidic channel connected to the inlet and into which a cell-containing fluid Qfc flows;
An interconnection channel consisting of a channel through which the concentrated cells Qc flow and a fluid channel through which the cells filtered through the enrichment process Qf flow;
Cell concentrator of the pneumatic drive method comprising a. The method according to claim 4,
A cell concentrator of a pneumatic drive method, characterized in that the lower end of the upper microfluidic channel is integrally formed with a right-angled internal channel through which only fluid can pass. The method according to claim 4,
The upper microfluidic channel is a pneumatically driven cell concentrator, characterized in that it is formed in a streamlined shape in which at least two bends are formed so that cells are concentrated while fluid is discharged through the internal channel. The method according to claim 1,
Cell concentrator has a multi-layer structure,
A fluid and pneumatic supply layer for external fluid connection and pneumatic connection by forming the inlet, outlet, and pneumatic connections;
A valve diaphragm layer for forming a valve membrane of the pneumatic valve;
A three-dimensional microfluidic channel network layer on which the microfluidic channel is formed; And
An additional sealing layer provided below the 3D microfluidic channel network layer;
Cell concentrator of the pneumatic drive method comprising a. The method according to claim 7,
The 3D microfluidic channel network layer includes an upper layer in which microfluidic channels are formed for injecting and discharging fluid containing cells,
The pneumatically driven cell concentrator, characterized in that it consists of a lower layer on which an interconnection channel is formed to form an interconnection structure between the pneumatic valves. A step in which a fluid containing cells is introduced through an inlet port;
When external compressed air is applied to the manure valve equipped with a valve diaphragm, the valve membrane (Valve diaphragm) is deformed to form a fluid channel formed on an inclined surface connecting the microfluidic channel and the hemispherical fluid chamber. As the exit surface is narrowed, the cells are blocked, and a surface where a fluid can be discharged is created (Sieve surface);
By turning off the fluid control valve (Vf) that flows only the fluid, and controlling the cell collection valve (Vc) that controls the passage through which the fluid containing the concentrated cells flows to obtain the cell concentrate (Qc), respectively. Concentrating the cells; And
Turn off (OFF) the external compressed air that was applied to the filter valve (Vs), and at the same time open the channel through which the concentrated cells (Qc) flow, and turn off the cell collection valve (Vc) to block the channels of the fluid (Qf). , By controlling the fluid control valve (Vf) to ON, to discharge the concentrated cells to the outside;
Cell concentration method using a pneumatically driven cell concentrator proceeding, including. The method according to claim 9,
A cell concentration method using a pneumatically driven cell concentrator, characterized in that by discharging a fluid through an embedded channel while the cells are concentrated, cells are deformed and prevent clogging of the channels. The method according to claim 9,
Cell concentration method using a pneumatically driven cell concentrator, characterized in that by controlling the pressure applied to the manure valve (Vs) according to the size of the cell, to control the deformation of the valve membrane, and concentrating cells of various sizes. A first step of applying a liquid resin to a first mold for making a hemispherical fluid chamber to a predetermined thickness;
A second step of curing the first mold coated with the liquid resin in the first step;
A third step of applying a liquid resin in a predetermined thickness to a second mold for forming a right-angled embedded channel;
A fourth step of curing the second mold coated with the liquid resin in the third step;
Fifth bonding of the first cured resin in which the air cavity is formed by removing the first mold from the first cured resin cured in the second step using atmospheric pressure plasma to the second cured resin cured in the fourth step step;
A sixth step of removing the second mold from the second cured resin and bonding the bonded cured resin to a glass substrate using atmospheric pressure plasma;
A seventh step of aligning the cured resin after applying a liquid resin to a third mold for manufacturing a lower layer of a three-dimensional microfluidic channel network layer;
An eighth step of curing the seventh step liquid resin;
A ninth step in which the air inside the air cavity, which is a closed space, expands, and the deformed reversed phase is replicated to the liquid resin as the resin film swells; And
A tenth step in which a microfluidic channel in which a right-angled embedded channel is formed is cured while the reverse phase of the deformation of the resin film is duplicated and cured;
Method of manufacturing a microfluidic channel of a cell concentrator of a pneumatically driven method consisting of. A first step of applying a liquid resin to a first mold for forming a hemispherical fluid chamber to a predetermined thickness;
A second step of curing the first mold coated with the liquid resin in the first step;
A third step of applying the liquid resin to the second mold in a predetermined thickness;
A fourth step of curing the second mold coated with the liquid resin in the third step;
A fifth step of bonding the first cured resin cured in step II to the second cured resin cured in step IV using atmospheric pressure plasma and then bonding the glass substrate to the glass substrate using atmospheric pressure plasma;
A sixth step of curing the liquid resin of the fifth step;
The air inside the air cavity, which is a closed space, expands, and as the resin film swells, the deformed reversed phase replicates to the liquid resin to make contact with the mold structure for fabricating the lower microfluidic channel network layer, resulting in a through structure. Forming a removing step; And
A step of completing the pneumatic valve structure by curing the through structure;
Method of manufacturing a pneumatic valve of a cell concentrator of a pneumatic drive method comprising a.

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