KR101433091B1 - Chematoxis Analysis Microfluidic Apparatus of bacteria, Production Method and Chematoxis Analysis Method of bacteria - Google Patents

Abstract

A microfluidic device for the analysis of the chemotaxis of bacteria, a method for producing the same, and a method for analyzing the chemotaxis of bacteria using the same. The microfluidic device for analyzing the chemotaxis of the bacteria includes a glass bottom substrate provided from the outside; And a PDMS upper substrate formed to be in contact with the glass lower substrate, wherein the upper substrate comprises: at least one cell culture chamber for culturing a plurality of cells; A bacteria injection port formed in the PDMS upper substrate and configured to inject bacteria provided from the outside into the upper substrate; Wherein the PDMS upper substrate has at least one collagen gel injection port into which a collagen gel provided from the outside is injected, wherein the PDMS upper substrate is connected to the cell culture chamber, the bacterial injection channel inlet, the bacteria extraction channel outlet, Are formed.

Description

TECHNICAL FIELD The present invention relates to a microfluidic device for analyzing the chemotaxis of bacteria, a method for producing the microfluidic device, and a method for analyzing the chemotaxis of the bacteria using the microfluidic device.

The present invention relates to a fl uidic device, and more particularly, to a fl uidic device in which a plurality of cells are co-cultivated on a single substrate, and an independent culture space capable of forming a co- A microfluidic device for analyzing the chemotaxis of bacteria for physically separating bacteria, a manufacturing method thereof, and a method for analyzing the chemotaxis of bacteria using the same.

Bacteria have the property of moving in the opposite direction of a substance or environment that moves or disgustes in the direction of a preferred substance or environment in the presence of a concentration gradient of a specific substance or environmental condition. The characteristic of these bacteria is called the dominant phenomenon, and the phenomenon of moving according to the gradient of the biochemical substance is called the chemotaxis.

Since the 19th century, bacteria have observed the chemotaxis of cancer tissues or cells, and efforts to treat cancer have continued. Despite these efforts, the direct cause of bacterial cancer detection has yet to be revealed. In order to use bacteria for therapeutic purposes, it is necessary to be able to identify the obvious cause of bacteria preferring cancer and to be prepared for the side effects caused by bacteria.

The reason bacteria prefer cancer is largely presumed to be the manifestation of the oxygen deficiency environment caused by the inadequate supply of cancer tissue and the biochemical substances that bacteria specifically secrete from cancer cells.

Cancer cells are unstable oxygen and nutrient supply due to abnormally rapid proliferation as a cancer tissue, resulting in a gangrene-deficient area in the tissue.

Bacteria that show chemotaxis to cancer tissues are usually known as anaerobic bacteria, so it is natural to assume that the chemotaxis of bacteria to cancer tissues is due to the oxygen deficiency environment inside the tissues.

However, cancer cells continue to secrete massive quantities of cancer-specific biochemical substances that are not normally expressed in normal cells for rapid proliferation as a tissue. If bacteria is the cause of cancer-causing biochemical matter, In order to establish the concentration gradient environment for the substances secreted in real time and to analyze the chemotaxis,

In addition, in order to confirm the directivity toward the cancer cell-specific substance, it is necessary to confirm the directivity of bacteria in an environment in which normal cells and cancer cells are cultured together under the same conditions. Therefore, conditions for simultaneous cultivation of two or more cells in one environment should be provided.

The most basic method for identifying chemotaxis is capillary analysis.

FIG. 1 is a view showing a method of analyzing the chemotaxis of bacteria according to the prior art, and FIG. 2 is a view showing a chemotaxis analysis method based on a microfluidic device according to the prior art.

As shown in FIG. 1, a capillary having a specific substance inserted therein is put into a bacterial solution, and a concentration gradient is generated through diffusion to analyze the chemotaxis of the bacteria.

However, this method is limited to the degree of understanding the tendency rather than the accurate analysis because the criterion which can judge the harmony is made through the qualitative method. It is also difficult to analyze a large number of samples at the same time.

Another proposed method for the analysis of the chemotaxis of bacteria is the microfluidic device published by Professor Jayaraman of the Chemical Engineering Department of Texas A & M University.

As shown in FIG. 2, two solutions for confirming the chemotaxis are injected through a gradient generator to cause a stable concentration gradient in the chemotaxis chamber.

Bacteria are injected at the top of the chemotaxis confirmation chamber to allow for the analysis of chemotaxis through movement within the chamber. This method is easy to analyze because it has all the components in one small substrate, but it requires continuous flow flow to form stable concentration gradient, and thus a large amount of culture solution is needed during the experiment. This continuous flow can also have unintended effects when analyzing bacteria and highly motile organisms.

Symbiotic cultures are usually a mixture of two or more cells, which is usually used to directly compare two or more cells in the same environment. However, if the cultures of the cells to be analyzed are different, such a symbiotic culture becomes difficult. All of these cells should be able to grow in a single culture medium, in which case conventional cultures may not be able to be used and the effects on conditions and materials that can only occur through normal culture should be excluded.

The problem to be solved by the present invention is to provide a real-time concentration gradient environment for biochemical substances generated by co-culturing a plurality of cells on a single substrate, to evaluate the chemotaxis of bacteria by a quantitative method, And a method for analyzing the chemotaxis of bacteria using the microfluidic device.

The microfluidic device for analyzing the chemotaxis of bacteria according to an embodiment of the present invention for solving the above problems is an independent culture space capable of creating a chemotactic environment in real time using materials secreted from cells, A collagen gel inlet for separation, and a channel through which bacteria can be injected.

According to an aspect of the present invention, there is provided a microfluidic device for analyzing the chemotaxis of bacteria, comprising: a glass bottom substrate provided from the outside; And a PDMS upper substrate formed to contact with the glass lower substrate, wherein the PDMS upper substrate comprises at least one cell culture chamber for culturing a plurality of cells; A bacteria inlet formed in the upper substrate and configured to inject bacteria provided from the outside into the upper substrate; And at least one collagen gel injection port into which a collagen gel provided from the outside is injected, wherein the upper substrate has fine flow channels connected to the cell culture chamber, the bacteria injection port, and the collagen gel injection port.

The collagen gel injection ports are formed in the same number as the number of the cell culture chambers.

Wherein the micro channel includes a main channel connected to the bacteria inlet port to move the bacteria; And a sub channel connected to the main channel and connected to the collagen gel injection port and the cell culture chamber, wherein the sub channel includes a cell culture chamber and a cell culture chamber, And a diffusion channel in which cell diffusion takes place.

And both ends of the diffusion channel are formed at an angle of at least 110 ° to less than 180 °.

According to an aspect of the present invention, there is provided a method of manufacturing a microfluidic device for analyzing the chemotaxis of bacteria, comprising the steps of: providing a glass bottom substrate from outside; And a second step of bonding the PDMS upper substrate on the glass lower substrate through an oxygen plasma treatment process after manufacturing a PDMS upper substrate from the outside.

In the second step, organic substances on the surface are removed with a Piranha solution in which sulfuric acid and hydrogen peroxide are mixed in a ratio of 4: 1 to a silicon wafer provided from the outside, and then a negative photosensitive agent SU-8 is applied using a spin coater An application step; After the application step, soft baking is performed to remove the organic solvent contained in the SU-8, and resin bonding of the negative photoresist is formed through exposure and post bake, followed by hard baking A pattern forming step of forming a fine flow path pattern on the silicon wafer; A PDMS substrate was produced by pouring a mixed solution of PDMS and a curing agent in a non-oil mixed solution on a silicon wafer having the microchannel pattern formed thereon, and then curing the PDMS substrate. Thereafter, a PDMS substrate for forming a cell culture chamber, a bacterial injection port and a collagen gel injection port An upper substrate generating step; And bonding the glass lower substrate and the PDMS upper substrate by oxygen plasma treatment.

The PDMS upper substrate forming step is a step of curing the mixed solution at a temperature of 70 ° C for 3 hours.

The bonding step is a step of bonding the PDMS upper substrate on the glass lower substrate by treating the oxygen plasma with a power of 200 W for 2 minutes and 30 seconds.

According to an aspect of the present invention, there is provided a method for analyzing the chemotaxis of bacteria using a microfluidic device for analyzing the chemotaxis of bacteria, comprising the steps of injecting a collagen gel into a collagen gel injection port and injecting collagen gel ; Culturing the cells in a cell culture chamber; Injecting a bacteria in which a fluorescent protein has been modified into a bacterial injection port; And analyzing the degree of chemotaxis of the bacteria diffused into the diffusion channel through the fluorescence intensity of the bacteria against the cell type located in the diffusion channel.

According to the present invention, it is possible to cultivate two or more kinds of cells in a single substrate for a long period of time using a conventional culture solution used in each independent space, thereby forming a real-time concentration gradient for secreted biochemical substances There is an advantage. In addition, it is possible to analyze more clearly and objectively through a quantitative method of comparing the degree of fluorescence of a plurality of samples.

Specifically, first, the collagen gel injected between the cell culture chamber and the bacterial injection channel forms a solid physical wall, thereby providing an independent culture space so that each culture solution is not mixed into the bacterial injection channel or the xenogeneic cell culture chamber.

Secondly, by using the porous nature of the collagen gel forming the wall, it is possible to analyze the chemotaxis of the bacteria by forming a real time concentration gradient in the bacterial channel through diffusion of various biochemical substances secreted from the cells for a long time.

Thirdly, since the analysis is carried out in the stationary flow, the chemotaxis of the bacteria can be performed without affecting the motility, and the use of the reagent such as the culture liquid can be reduced to a few hundred μl or less.

Fourth, by comparing the fluorescence intensity of bacteria after a certain period of time using the bacteria in which green fluorescent protein (GFP) is formulated, objective and quantitative analysis is possible.

Fifth, the manufacturing process can be made simple and low-cost by using a conventional soft lithography method.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a method for analyzing the chemotaxis of bacteria according to the prior art.
FIG. 2 is a diagram illustrating a method for analyzing chemotaxis based on a microfluidic device according to the prior art.
3 is a plan view of an upper substrate of a microfluidic device for analyzing the chemotaxis of bacteria according to the present invention.
4 is a perspective view of a microfluidic device for analyzing the chemotaxis of bacteria according to the present invention.
FIG. 5 is a partial perspective view of a microfluidic device in which a chemotaxis analysis region of a bacteria belonging to the present invention is enlarged. FIG.
FIG. 6 is a plan view showing a chemotaxis analysis region of a bacteria belonging to the present invention. FIG.
FIG. 7 is an enlarged perspective view of the end portion of the collagen gel injection region of FIG. 6. FIG.
8A is an exemplary diagram for representing the theoretical capillary pressure in the expansion channel according to the channel shape coefficient and the contact angle.
8B is a graph showing the theoretical capillary pressure shown in FIG. 8A.
FIG. 9 is a flowchart illustrating a method of manufacturing a microfluidic device for analyzing the chemotaxis of bacteria according to an embodiment of the present invention.
10 is a flowchart showing the second step shown in Fig. 9 in more detail.
11 is a flow chart showing a manufacturing process of a microfluidic device for analyzing the chemotaxis of bacteria according to the present invention.
FIG. 11 is a schematic diagram showing a procedure for analyzing the fluorescence Salmonella chemotaxis of liver cancer and normal cells according to an embodiment of the present invention. FIG.
FIG. 12 is a table showing a method for analyzing the salmonella chemotaxis result according to an embodiment of the present invention.

Specific structural and functional descriptions of embodiments according to the concepts of the present invention disclosed in this specification or application are merely illustrative for the purpose of illustrating embodiments in accordance with the concepts of the present invention, The examples may be embodied in various forms and should not be construed as limited to the embodiments set forth herein or in the application.

Embodiments in accordance with the concepts of the present invention can make various changes and have various forms, so that specific embodiments are illustrated in the drawings and described in detail in this specification or application. It should be understood, however, that the embodiments according to the concepts of the present invention are not intended to be limited to any particular mode of disclosure, but rather all variations, equivalents, and alternatives falling within the spirit and scope of the present invention.

The terms first and / or second, etc. may be used to describe various components, but the components should not be limited by the terms. The terms may be referred to as a first component second component only for the purpose of distinguishing one component from another component, for example without departing from the scope of the right according to the concept of the present invention, The two components can also be named as the first component.

It is to be understood that when an element is referred to as being "connected" or "connected" to another element, it may be directly connected or connected to the other element, .

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprises ",or" having ", and the like, are intended to specify the presence of stated features, integers, steps, operations, elements, , Steps, operations, elements, parts, or combinations thereof, as a matter of principle, without departing from the spirit and scope of the invention.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the preferred embodiments of the present invention with reference to the accompanying drawings. Hereinafter, the present invention will be described in more detail with reference to the drawings.

FIG. 3 is a top view of an upper substrate of a microfluidic device for analyzing the chemotaxis of bacteria according to the present invention, FIG. 4 is a perspective view of a microfluidic device for analyzing the chemotaxis of bacteria according to the present invention, FIG. 6 is a plan view showing a chemotaxis analysis region of a bacteria belonging to the present invention, and FIG. 7 is a cross-sectional view of the microfluidic device of FIG. FIG.

As shown in FIG. 3, the microfluidic device 1000 for analyzing chemotaxis of bacteria according to the present invention can be used as an independent culture space capable of creating a chemotactic environment in real time using materials secreted from cells and the like, A collagen gel injection port for separating the bacteria into the bacteria, and a channel through which bacteria can be injected.

More specifically, the apparatus 1000 includes a glass bottom substrate 200 and a PDMS top substrate 100.

The PDMS upper substrate 100 is formed to be in contact with the glass lower substrate 200.

The PDMS upper substrate 200 includes bacterial injection ports 120 and 130, a collagen gel injection port 140, and a cell culture chamber 110.

The cell culture chamber 110 cultivates various kinds of cells.

The bacteria injection ports 120 and 130 are formed in the PDMS upper substrate 100 and are formed to inject bacteria supplied from the outside.

The collagen gel injection port 140 is injected with collagen gel provided from the outside, and at least one or more collagen gel injection holes 140 are formed in the PDMS upper substrate 100.

At this time, the PDMS upper substrate 100 has a micro channel for connecting the cell culture chamber 110, the bacteria injection ports 120 and 130, and the collagen gel injection port 140.

The PDMS upper substrate 100 and the glass lower substrate 200 are bonded through an oxygen plasma treatment process.

The collagen gel injection ports 140 are formed in the same number as the number of the cell culture chambers 110.

The micro channel includes a main channel connected to the bacteria injection ports 120 and 130 and connected to the main channel and a sub channel connected to the collagen gel injection port 140 and the cell culture chamber 110 And the sub channel may include a diffusion channel 141 in which cell diffusion occurs depending on the chemotaxis of cells in the cell culture chamber 110 and bacteria moving in the main channel.

Both ends of the diffusion channel 141 may be formed at an angle of at least 110 ° to less than 180 °.

More specifically, the angle of the diffusion channel is preferably 110 °, which is intended to passively cause the flow of the collagen gel to stop to form a controlled-type collagen gel wall.

8A is an exemplary view for expressing the theoretical capillary pressure in the extended channel according to the channel shape coefficient and the contact angle, and FIG. 8B is a graph showing the theoretical capillary pressure shown in FIG. 8A.

Referring to FIG. 8A, the progress of the fluid in the shape in which the channel having a constant height is expanded is shown. The pressure of the capillary flow at the expansion point can be expressed by Equation 1 below.

[Equation 1]

Where? Is the contact angle,? Is the channel extension angle,? Is the surface tension coefficient, and? Is the radius of curvature of the fluid interface.

The capillary pressure Pn can be rewritten as: < EMI ID = 2.0 >

&Quot; (2) "

When the contact angle is θ and θi, the pressure is as follows. At this time, if the capillary pressure Pn has a negative value, it is understood that the capillary flow stops at the expansion point and the flow proceeds when the capillary pressure Pn has a positive value.

Referring to FIG. 8B, it can be seen that as the width of the capillary is small, the capillary tube has a large stopping pressure and a maximum stopping pressure appears at a certain extension angle when the height is constant. Here, the width h of the capillary is 100 μm, The expansion angle [theta] is 70 [deg.]).

Also, even when the contact angle of the channel is different, the change of the stopping pressure can easily be checked using Equation (1).

That is, the apparatus 1000 of the present invention is an apparatus for quantitatively analyzing the chemotaxis of bacteria to different biochemical substances expressed by culturing two or more kinds of cells, and comprises a PDMS material upper substrate 100 And a lower substrate 200 made of a glass material. All the components for the chemotaxis analysis are designed as a monolayer channel (for example, a microchannel) in the PDMS upper substrate 100.

The PDMS upper substrate 100 is bonded to the lower glass substrate 200 and is processed with oxygen plasma at a constant power for a predetermined time so as to be integrated. At this time, it is preferable that the bonding is performed after treatment with oxygen plasma 200 W for 2 minutes and 20 seconds.

The PDMS upper substrate 100 is provided with a cell culture chamber 110 in which a plurality of cells can be cultured, bacterial injection ports 120 and 130 into which bacteria can be injected, collagen A gel inlet 140 is provided. These components can be fabricated through a very simple MEMS process because they are composed of one channel without additional processing.

The process for analyzing the chemotaxis of bacteria using a microfluidic device for analyzing the chemotaxis of bacteria according to the present invention is as follows. Two or more cell lines using different culture media in a separate space formed by injection of collagen gel into the microfluidic device are cultured for a certain period of time.

At this time, in the design of the injection port of the collagen gel, the shape of the channel was adjusted so that the collagen gel 141 could be filled only at the designated position. Referring to FIG. 6, the phenomenon that the flow stops when the shape of the channel sharply expands to 90 ° or more in the micro-unit flow dominated by the capillary force and the surface tension, is used for collagen cell culture chamber- At both ends of the gel injection part, the shape of the channel is designed so that the width thereof is rapidly expanded, and the flow of the gel is manually stopped when injecting, thereby inducing the formation of the controlled collagen gel wall. At this time, it is desirable that the angle of expansion of the channel is designed to be not less than 110 ° and less than 180 ° for stable flow control (see FIG. 7).

While the cells are being cultured, the biochemical substances secreted from the cells are spread into the collagen gel while the bacterial injection center channel is not filled.

After a period of time, bacteria are injected into the central channel to analyze the chemotaxis of the bacteria using a concentration gradient formed by the diffusion of biochemical substances at the end of the collagen gel connected to the center channel.

At this time, for the quantitative analysis, the bacteria to be used are obtained by modifying a substance such as green fluorescent protein (GFP) to determine the number of bacteria that have been put in a specific direction in the flammability analysis, (See FIG. 6), and the fluorescence brightness is quantitatively compared with the graphic software to analyze the fluorescence.

FIG. 9 is a flow chart showing a method of manufacturing a microfluidic device for analyzing the chemotaxis of bacteria according to an embodiment of the present invention, FIG. 10 is a flowchart showing the second step shown in FIG. 9 in more detail, 11 is a flowchart showing a manufacturing process of a microfluidic device for analyzing the chemotaxis of bacteria according to the present invention.

9 to 11, a method (S100) of manufacturing a microfluidic device for analyzing the chemotaxis of bacteria according to an embodiment of the present invention includes a first step (S110) and a second step (S120) do.

The first step (S110) is a step of providing a glass bottom substrate from the outside.

In the second step (S120), the PDMS upper substrate is manufactured from the outside, and then the PDMS upper substrate is bonded to the glass lower substrate through an oxygen plasma processing process.

More specifically, the second step S120 includes an applying step S121, a pattern forming step S122, a PDMS upper substrate producing step S123, and a bonding step S124.

In the coating step (S121), organic substances on the surface are removed with a Piranha solution in which sulfuric acid and hydrogen peroxide are mixed in a ratio of 4: 1 to a silicon wafer provided from the outside, and then a negative photoresist SU-8 It may be a step of applying.

In the pattern formation step S122, after the application step, soft baking is performed to remove the organic solvent contained in the SU-8, and resin bonding of the negative photoresist is formed through exposure and post bake , And a hard baking process to form a fine flow path pattern on the silicon wafer.

In the PDMS upper substrate formation step (S123), a mixture solution of PDMS and a curing agent in a ratio of 10: 1 is poured on a silicon wafer having the microchannel pattern formed thereon, and then cured to form a PDMS substrate. A bacterial injection port and a collagen gel injection port.

The bonding step (S124) may be a step of bonding the lower glass substrate and the PDMS upper substrate by oxygen plasma treatment.

The PDMS upper substrate forming step S123 may be a step of curing the mixed solution at a temperature of 70 DEG C for 3 hours, and the bonding step (S124) may be a step of heating the oxygen plasma to 200 DEG C for 2 minutes For 30 seconds to bond the PDMS top substrate onto the glass bottom substrate.

FIG. 12 is a schematic view showing a fluorescence Salmonella assimilation analysis process of hepatic cancer and normal cells according to an embodiment of the present invention, and FIG. 13 is a table showing a method of analyzing Salmonella chemotaxis result analysis according to an embodiment of the present invention.

12, cancer cells and hepatic normal cells are cultured for a predetermined time in the microfluidic device cell culture chamber 110 provided with an independent culture space due to the collagen gel injected through the collagen gel injection port 140 Fluorescent bacteria injected through the central bacterial injection port 120 were injected and analyzed.

Referring to FIG. 13, fluorescence photographs were taken at a predetermined exposure time (200 ms) in the analysis area (FIG. 6) and numerical comparison of the brightness of fluorescence to this part was performed. The chemotaxis of Korean bacteria can be analyzed.

Therefore, according to the present invention, the cells and the culture liquid injected into the cell culture chamber can be cultured independently in a space for a long time due to the collagen gel which forms a solid wall, and the biochemical substances generated thereby are diffused into the porous collagen gel .

When the PDMS upper substrate and the glass substrate are integrated, two or more kinds of cells are independently cultured in each cell culture chamber, and when the bacteria solution is injected into the central bacterial injection channel, the biochemical material diffused from the end of each collagen gel It will analyze the chemotaxis of bacteria.

For more objective quantitative analysis, we use green fluorescent protein (GFP) -modified bacteria to determine the degree of chemotaxis by measuring the brightness of fluorescence caused by bacteria in each location.

According to the present invention, it is possible to cultivate two or more kinds of cells in a single substrate for a long period of time using a conventional culture solution used in each independent space, thereby forming a real-time concentration gradient for secreted biochemical substances There is an advantage. In addition, it is possible to analyze more clearly and objectively through a quantitative method of comparing the degree of fluorescence of a plurality of samples.

Specifically, first, the collagen gel injected between the cell culture chamber and the bacterial injection channel forms a solid physical wall, thereby providing an independent culture space so that each culture solution is not mixed into the bacterial injection channel or the xenogeneic cell culture chamber.

Secondly, by using the porous nature of the collagen gel forming the wall, it is possible to analyze the chemotaxis of the bacteria by forming a real time concentration gradient in the bacterial channel through diffusion of various biochemical substances secreted from the cells for a long time.

Thirdly, since the analysis is carried out in the stationary flow, the chemotaxis of the bacteria can be performed without affecting the motility, and the use of the reagent such as the culture liquid can be reduced to a few hundred μl or less.

Fourth, by comparing the fluorescence intensity of bacteria after a certain period of time using the bacteria in which green fluorescent protein (GFP) is formulated, objective and quantitative analysis is possible.

Fifth, the manufacturing process can be made simple and low-cost by using a conventional soft lithography method.

As described above, the microfluidic device for analyzing the chemotaxis of bacteria, the method for producing the same, and the method for analyzing the chemotaxis of bacteria using the same can be variously modified and take various forms. It is to be understood, however, that the invention is not to be limited to the specific forms thereof, which come within the scope of the appended claims, including all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims .

100: PDMS upper substrate 110: Multi-cell culture chamber
120, 130: Bacteria injection port 140: Collagen gel injection port
141: diffusion channel 200: glass bottom substrate
1000: Fluidic device

Claims (10)

delete A glass lower substrate provided from the outside;
And an upper substrate formed to be bonded onto the lower glass substrate,
In the upper substrate,
At least two cell culture chambers for culturing a plurality of cells;
A bacteria inlet formed in the upper substrate and configured to inject bacteria provided from the outside into the upper substrate;
And at least two collagen gel injection ports into which a collagen gel provided from the outside is injected,
Wherein the upper substrate comprises:
A micro channel for connecting the cell culture chamber, the bacterial injection port, and the collagen gel injection port is formed,
The fine flow path,
A main flow channel connected to the bacteria inlet port to move the bacteria; And
And a sub channel connected to the main channel and connected to the collagen gel inlet and the cell culture chamber,
The sub-
And a diffusion channel in which cell diffusion occurs depending on the chemotaxis of cells in the cell culture chamber and bacteria moving in the main channel,
The angle between the extension line extending the end of the diffusion flow passage and the main flow passage is at least 110 but less than 180,
Wherein the diffusion channel is filled with collagen gel injected from the injection port of the collagen gel, and the collagen gel is not introduced into the main flow channel.
3. The method of claim 2,
In the collagen gel injection port,
Wherein the microfluidic device is formed in the same number as the number of the cell culture chambers.
delete delete A first step of providing a glass bottom substrate from outside; And
And a second step of bonding the PDMS upper substrate to the lower glass substrate through an oxygen plasma treatment process after the PDMS upper substrate is manufactured from the outside. A method of manufacturing a microfluidic device.
The method according to claim 6,
The second step comprises:
A step of applying a negative photosensitive agent SU-8 using a spin coater after removing organic substances on the surface with a Piranha solution in which sulfuric acid and hydrogen peroxide are mixed in a ratio of 4: 1 to a silicon wafer provided from the outside;
After the application step, soft baking is performed to remove the organic solvent contained in the SU-8, and resin bonding of the negative photoresist is formed through exposure and post bake, followed by hard baking A pattern forming step of forming a fine flow path pattern on the silicon wafer;
A PDMS substrate was produced by pouring a mixed solution of PDMS and a curing agent in a non-oil mixed solution on a silicon wafer having the microchannel pattern formed thereon, and then curing the PDMS substrate. Thereafter, a PDMS substrate for forming a cell culture chamber, a bacterial injection port and a collagen gel injection port An upper substrate generating step; And
And bonding the lower glass substrate and the upper PDMS substrate by oxygen plasma treatment to bond the lower glass substrate to the PDMS upper substrate.
8. The method of claim 7,
The PDMS upper substrate generating step may include:
And curing the mixed solution at a temperature of 70 캜 for 3 hours. 2. The method according to claim 1, wherein the microfluidic device is a microfluidic device.
8. The method of claim 7,
In the joining step,
Treating the oxygen plasma at a power of 200 W for 2 minutes and 30 seconds to bond the PDMS top substrate onto the glass bottom substrate.
A collagen gel injecting step of injecting a collagen gel into the injection port of the collagen gel to fill the diffusion channel;
Culturing the cells in a cell culture chamber;
Injecting a bacteria in which a fluorescent protein has been modified into a bacterial injection port; And
A microfluidic device for analyzing the chemotaxis of bacteria according to claim 2, comprising a step of analyzing the degree of chemotaxis of bacteria diffused into the diffusion channel through the fluorescent brightness of bacteria to a cell type located in the diffusion channel Method for the analysis of chemotaxis using.

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