JP4672511B2 - Micro reactor - Google Patents

Description

  The present invention relates to a microreaction apparatus for allowing a reagent to act on a solid sample held in a minute reaction chamber, for example, introducing a drug selectively into a part of a biological sample such as a cell tissue and observing the reaction. Thus, the present invention relates to a microreaction apparatus such as a microcell analysis apparatus for analyzing the function of a cell tissue and the utility of a drug.

So far, microfluidic devices fabricated using MEMS (Micro Electro Mechanical System) technology for applying drugs to cells and measuring their reactions have been reported ( (See Non-Patent Documents 1 to 4.)
By using these devices, analysis of a small amount of cells and high-throughput screening of drugs can be realized.

These conventional devices can be classified into two types. One is a device intended to analyze the reaction of each cell, as in Non-Patent Documents 1 and 2, and the other is a function of multiple cells as in Non-Patent Documents 3 and 4, such as tissue. It is a device intended to analyze the reaction of cells.
A. Tixier, et al. Micro Total Analysis Systems 2000, pp123-126 (2000) Kwang-Seok Yun, et al. Micro Total Analysis Systems 2002, pp652-654 (2002) Sensors and Actuators A, Vol.114, pp129_134 (2004) Kwang-Seok Yun, et al. Micro Total Analysis Systems 2003, Vol. 1, pp861-864 (2003)

The two types of conventional microfluidic devices for cell analysis described above have different advantages and problems.
First, in a device intended to analyze the reaction of one cell, a drug can be selectively reacted to one cell, but the function as a cell tissue cannot be analyzed. For example, since a liver cell or the like cannot express its original function with a single cell, it is difficult to analyze the function that is actually expressed in vivo.

  On the other hand, in a device intended to analyze the reaction of a plurality of cells, it is difficult to selectively react a drug with a specific cell or site. When a chemical solution is introduced into the reaction chamber, the chemical solution spreads throughout the reaction chamber, so that the chemical solution reacts with the entire tissue. The interaction of cells as a tissue is considered to be important because one cell reacts with a reagent, the substance produced by that cell acts on another cell, and the cell produces a new substance. Yes. Therefore, in order to analyze the detailed function of cellular tissue, it is important to elucidate the path (path) of the interaction. To that end, it is necessary to selectively apply to specific cells or parts of cellular tissue. It is necessary to react the drug.

Such a situation is not limited to the case where the measurement target is a cell, but generally exists when another solid sample is the target.
Accordingly, an object of the present invention is to provide a microreaction apparatus capable of selectively reacting a drug with a part of a plurality of solid samples in a reaction chamber.

  The microreaction apparatus of the present invention is formed in a cylindrical shape having an upper surface and a bottom surface, and is provided with a reaction chamber for accommodating a solid sample at the bottom inside, and the reaction chamber is formed on the upper surface of the reaction chamber. And a plurality of buffer inlets for introducing a buffer fluid into the reaction chamber, and a plurality of liquid outlets for discharging the liquid in the reaction chamber to the outside at the bottom corner. It is characterized by this.

It is preferable that the reagent introduction port is arranged at the center of the upper surface of the reaction chamber, and three or more buffer introduction ports are arranged so as to surround the reagent introduction port. More preferably, the buffer inlets are arranged at equal intervals on one circumference centered on the reagent inlet.
It is preferable that three or more liquid discharge ports are provided. More preferably, the liquid discharge ports are arranged at equal intervals at the bottom corner of the reaction chamber.

A preferable example of the shape of the reaction chamber is a cylindrical shape.
In a preferred example of the reaction chamber, the bottom has a double structure comprising a bottom surface and a bottom plate that is spaced from the bottom surface and holds the sample, and the bottom plate has a plurality of holes smaller than the sample to be held. The liquid discharge port is provided below the bottom plate. In this case, the reagent and the buffer fluid introduced into the reaction chamber are discharged from the liquid discharge port through the hole in the bottom plate.

  In the method of using the microreaction apparatus of the present invention, a plurality of samples are held at the bottom of the reaction chamber, and are introduced from the reagent inlet by adjusting the flow rate of the buffer fluid introduced from each of the buffer inlets. The flow direction of the reagent is controlled to selectively act on a part of the plurality of samples.

The sample held in the reaction chamber by the microreaction apparatus of the present invention is not particularly limited, but a preferred example is a living cell.
The microreaction apparatus of the present invention may further include a buffer fluid introduction mechanism that is connected to each of the buffer introduction ports and can adjust the flow rate of the introduced buffer fluid.

FIG. 1 schematically shows the configuration of the microreaction apparatus of the present invention. 1A is a schematic perspective view, and FIG. 1B is a cross-sectional view taken along the line AA.
The reaction chamber 2 is formed in a cylindrical shape having an upper surface 4 and a bottom surface in a substrate, and a solid sample 8 is accommodated in an inner bottom portion 6. The upper surface 4 is provided with a reagent introduction port 10 for introducing a reagent into the reaction chamber 2 and a plurality of buffer introduction ports 12 for introducing a buffer fluid into the reaction chamber 2. Further, a sample inlet 13 is provided in the upper part of the reaction chamber for introducing a sample to be analyzed. The bottom 6 is provided with a plurality of liquid discharge ports 14 at the corners for discharging the liquid in the reaction chamber 2 to the outside.

  The number and position of the reagent introduction ports are not particularly limited, but here, one is arranged in the center of the upper surface. The number and position of the buffer introduction ports 12 are not particularly limited, but here, four buffer introduction ports 12 are arranged so as to surround the reagent introduction port 10 arranged at the center of the upper surface. The bottom structure of the reaction chamber 2 is not limited to this, but here, the bottom 6 has a double structure comprising a bottom surface 6a and a bottom plate 6b that is spaced from the bottom surface 6a and holds the sample 8. The bottom plate 6b has a mesh structure in which a plurality of holes smaller than the sample 8 to be held are formed, and the sample 8 such as a cell to be analyzed is held on the bottom plate 6b for reaction and culture. . The liquid discharge port 14 is provided below the bottom plate 6b, and the reagent and the buffer fluid introduced into the reaction chamber 2 are discharged from the liquid discharge port 14 through the holes of the bottom plate 6b. .

However, the present invention includes a reaction chamber having no bottom plate 6b and a sample 8 held on the bottom surface 6a.
Here, the sample introduction port 13 is provided in the upper part of the reaction chamber. However, if the sample to be analyzed is an object smaller than the reagent introduction port 10 or the buffer introduction port 13, the sample is introduced into the reaction chamber from any of the introduction ports in advance. Since it can be introduced, a sample inlet is not necessarily required.

  When the case where such a microreaction apparatus is used for cell culture is taken up, a plurality of cells 8 are held as samples in the reaction chamber 2, and a reagent is supplied from the reagent inlet 10 and a buffer solution is simultaneously supplied from the buffer inlet 12. Introduce. As shown in FIG. 1B, the reagent flow 10a reaches the reaction chamber bottom 6, that is, the cell 8 to be analyzed, in a state surrounded by the buffer flow 12a. Since the reagent flow 10a flows in a state surrounded by the buffer flow 12a, the reagent does not spread over the entire reaction chamber 2, and the reagent flow reaches only a part of the reaction chamber bottom 6, that is, a part of the plurality of cells 8. To do.

The direction of the flow of the reagent flow 10a can be controlled by adjusting the flow rate balance of the buffer flow 12a introduced from a plurality of (in this case, four) buffer introduction ports 12.
As described above, the buffer inlet 12 is arranged around the reagent inlet 10, and the buffer flow 12a is flowed at a flow rate controlled so as to surround the reagent flow 10a, thereby allowing a plurality of cells in the reaction chamber 2 to flow. The reagent can be reacted with a part of 8.
Such an operation is not limited to the case where the sample is a cell, but is the same when the sample is another solid sample.

  In the microreaction apparatus of the present invention, a reagent introduction port and a plurality of buffer introduction ports are provided on the upper surface of the reaction chamber, and a plurality of liquid discharge ports are provided at the bottom corner, so that a plurality of The reagent can be selectively reacted with a specific part of each solid sample. Thereby, for example, a chemical solution can be reacted with a specific part of a cell tissue, and the reaction can be measured.

By arranging the reagent introduction port in the center of the upper surface and arranging three or more buffer introduction ports so as to surround the reagent introduction port, the direction control of the reagent flow by the buffer flow can be performed in a wide range.
Furthermore, if the buffer inlets are arranged at equal intervals on one circumference centered on the reagent inlet, the reagent flow direction control by the buffer flow can be performed uniformly over a wide range.

By setting the number of the liquid discharge ports to three or more, the direction control of the reagent flow by the buffer flow can be performed in a wide range.
Furthermore, if the liquid discharge ports are arranged at equal intervals in the bottom corner, the reagent flow direction control by the buffer flow can be performed uniformly over a wide range.
If the reaction chamber has a cylindrical shape, the direction control of the reagent flow by the buffer flow becomes easy.

If the bottom structure is a dual structure consisting of a bottom surface and a perforated bottom plate spaced from the bottom surface, the reagent and the buffer fluid are discharged from the liquid outlet through the holes in the bottom plate. It becomes easy to selectively supply a reagent to a part of the held sample, and the controllability of the flow of the fluid reagent in the reaction chamber can be improved.
If this reaction apparatus is further provided with a buffer fluid introduction mechanism, reaction control by this reaction apparatus can be easily performed.

Embodiments of the present invention will be described with reference to the drawings.
FIG. 2 shows a microreactor of one embodiment. (A) is a schematic perspective view, (B) is a top view, and (C) is a bottom view.
Here, a cell is measured as a sample. A reaction chamber 2 in which a reagent is reacted with the cell 8 to perform analysis is a cell culture chamber, and has a cylindrical shape. A reagent inlet 10 and eight buffer inlets 12 are arranged on the upper surface 4 of the reaction chamber 2 so as to surround it. The reagent inlet 10 is arranged at the center of the upper surface 4, and the eight buffer inlets 12 are arranged at equal intervals on one circumference centered on the reagent inlet 10. A sample inlet for introducing a sample to be analyzed is provided in the upper part of the reaction chamber.

On the other hand, eight liquid outlets 14 for discharging the reagent and the buffer are arranged at the bottom 6 of the reaction chamber 2. The liquid discharge ports 14 are arranged at equal intervals along the circular corner of the bottom 6.
A plurality of cells 8 to be analyzed are introduced into the reaction chamber 2 and held on the bottom surface of the reaction chamber 2.

  Reagents for reacting with cells are introduced from the reagent introduction port 10, and at the same time, buffers are introduced from the respective buffer introduction ports 12. At this time, the flow direction of the reagent in the reaction chamber 2 can be controlled by adjusting the buffer flow rate balance supplied from each buffer inlet 12.

  For introduction of the reagent from the reagent introduction port 10 and introduction of the buffer from the buffer introduction port 12, the flow rate may be controlled independently using a liquid feed pump. In addition, the container containing the reagent is connected to the reagent introduction port 10 via a tube, the container containing the buffer is connected to the buffer introduction port 12 via a tube, and the height for holding these containers is adjusted. You may make it control flow volume by.

  The reagent introduced from the reagent introduction port 10 reaches a part of the cells 8 on the bottom surface of the reaction chamber 2 while being surrounded by the buffer flow. Since the flow direction of the reagent can be controlled by controlling the buffer flow rate from each buffer flow inlet 12, it is possible to control the position on the bottom surface where the reagent reaches, that is, the target. The reagent can selectively reach some cells 8.

  At this time, the flow of the reagent and the buffer discharged from the reaction chamber 2 is also an important factor for determining the direction of the reagent flow. For example, when there is only one liquid discharge port 14 on the bottom surface of the reaction chamber 2, the buffer and the reagent introduced into the reaction chamber 2 always flow to the one liquid discharge port 14, so the buffer flow rate should be adjusted. Even if the flow of the reagent introduced from the reagent introduction port 10 is controlled by the above, it eventually reaches the liquid discharge port 14 at one place. As a result, the controllability of the reagent flow by the buffer flow is greatly impaired. However, in this embodiment, the liquid discharge ports 14 are arranged radially at the bottom corners of the reaction chamber 2 to eliminate such drawbacks.

FIG. 3 shows the result of computer analysis of the reagent introduction in this example. Coventor's analysis tool, Coventor Ware, was used for computer analysis.
As analysis conditions, rhodamine B aqueous solution was used as a reagent, and physical properties of pure water were used as a buffer.

(A) shows the outer shape of the reaction chamber 2. An example of the dimensions of each part is shown. The reaction chamber 2 is cylindrical with a diameter of 1000 μm and a height of 140 μm, and the reagent inlet 10 has a radius of 20 μm. The buffer inlet 12 has a radius of 50 μm, and the distance between the reagent inlet 10 and the buffer inlet 12 is 150 μm.
The analysis results in that case are shown in (B) and (C). (B) shows the reagent concentration distribution on the bottom surface of the reaction chamber 2, and (C) shows the reagent concentration distribution on the cross section of the reaction chamber 2.

From the result of (B), it can be seen that the reagent reaches only a part of the bottom surface of the reaction chamber 2. From the result of (C), it can be seen that the flow direction of the reagent flow rate is biased from the center of the reaction chamber 2 to the right side in the figure due to the flow rate difference of the buffer flow.
From these results, it can be seen that the reagent can selectively reach a part of the bottom surface of the reaction chamber 2 by using the reaction apparatus of this example.

  The reaction apparatus of this example was produced with the dimensions shown above, and the bottom surface was made of transparent glass, and the bottom surface was observed with a fluorescence microscope (and confocal fluorescence microscope) 20 as shown in FIG. Rhodamine B aqueous solution was used as a reagent, and pure water was used as a buffer.

  The flow of the reagent on the bottom surface can be controlled in an arbitrary direction by adjusting the flow rate of the buffer flow as shown in FIG. Further, the flow of the reagent in the depth direction is also biased to one side as shown in FIG. Thus, the simulation results are in good agreement with the results of actually producing the reactor and the computer analysis shown in FIG.

  The observation system as shown in FIG. 4 uses, for example, fluorescence anisotropy, eg, fluorescence resonance energy transfer (FRET) or fluorescence anisotropy analysis, to show how proteins are produced by cells. Thus, it can be used as a measuring device that can be observed in real time by a confocal fluorescence microscope.

As another embodiment of the present invention, a structure as shown in FIG. 7, which is different from the structure described above, can be given. The embodiment of FIG. 7 is the structure of the reactor of FIG. 1 described as schematically illustrating the present invention.
2 is different from the embodiment of FIG. 2 in that the bottom 6 of the reaction chamber 2 has a bottom structure 6a and a bottom plate 6b that is spaced from the bottom 6a and holds a sample 8 such as a cell. 6b is a mesh structure having a large number of pores smaller than the sample 8 to be held. The liquid discharge port 14 is formed below the bottom plate 6b.

  Although the structure described above includes one reagent introduction port 10, as another embodiment, a plurality of types of reagent introduction ports 10 can be provided to introduce a plurality of types of reagents into the reaction chamber 2. Furthermore, by arranging the buffer inlets 12 around each reagent inlet 10, it is possible to control the direction of each reagent flow.

  When this reaction apparatus is used as a cell culture chamber, the reagent and the buffer supplied to the reaction chamber 2 pass through the pores of the bottom plate 6b and are discharged downward from the bottom plate 6b. The sample cells 8 to be analyzed are cultured on the bottom plate 6b having a mesh structure at the bottom of the culture chamber. By using this structure, the flow of the reagent and the buffer is not easily affected by the culture chamber bottom surface 6a, so that the controllability of the flow of the reagent can be further improved.

Next, the manufacturing method of the embodiment of FIG. 2 will be described with reference to FIG.
The reaction chamber 2, the reagent inlet 10 and the buffer inlet 12 provided on the upper surface thereof, and the liquid outlet 14 provided at the bottom of the reaction chamber 2 are formed on the silicon substrate by photolithography and ICP-RIE (inductively coupled plasma type reactivity). (Ion etching).
(A) A silicon substrate 30 having a thickness of 200 μm is used, and a silicon oxide film 32 serving as a mask for dry etching is formed on both surfaces thereof to a thickness of 1 μm by thermal oxidation.

(B) A resist pattern is formed on the silicon oxide film 32 by photolithography using a double-sided aligner, and the silicon oxide film 32 is patterned by oxide film etching using hydrofluoric acid using the resist pattern as a mask. As a result, an oxide film pattern 32a (see FIG. (B-1) for a plan view) that determines the shape of the reagent inlet and the buffer inlet is formed on the upper surface of the silicon substrate 30, and the reaction chamber shape is formed on the lower surface of the silicon substrate 30. Then, an oxide film pattern 32b (see FIG. (B-2) for a plan view) for forming the liquid discharge port 14 and a flow path connected thereto is formed.
(C) A resist pattern 34 (see FIG. (C-1) for a plan view) for forming a reaction chamber shape is formed on the lower surface of the silicon substrate by photolithography using a double-sided aligner.

  (D) Using the photoresist pattern 34 on the lower surface side of the silicon substrate 30 as a mask, the silicon substrate 30 is dry-etched to a depth of 50 μm using ICP-RIE to form a part of the reaction chamber 2.

  (E) After removing the photoresist 34 on the lower surface of the silicon substrate with a mixed solution of sulfuric acid and peroxygenated water, the silicon substrate 30 is made to be ICP− using the oxide film pattern 32a on the upper and lower surfaces of the silicon substrate 30 as a mask. Dry etching is performed using RIE, and a reagent inlet 10 and a buffer inlet 12 are formed on the upper surface of the substrate, and a liquid outlet 14 is formed on the lower surface of the substrate. The etching depth is 50 μm.

(F) The silicon oxide films 32 on both sides of the silicon substrate 30 are removed.
(G) A glass substrate 36 is anodically bonded to the lower surface of the silicon substrate 30 thus formed. As the glass substrate to be anodically bonded to the silicon substrate, borosilicate glass such as Pyrex glass (registered trademark) is preferable.

The bonding of the silicon substrate and the glass substrate is not limited to the above method. Bonding with hydrofluoric acid may be performed in a state where the silicon oxide film 32 remains on both surfaces of the silicon substrate 30 or after the silicon oxide film is formed again. The type of glass substrate in this case is not particularly limited, and various types of glass such as quartz glass can be used.
Furthermore, you may join a silicon substrate and a glass substrate using an adhesive agent.

A method for producing the reactor of the embodiment of FIG. 7 will be described with reference to FIG.
(A) Similar to the manufacturing method of FIG. 8, a silicon substrate 30 is used, and a silicon oxide film 32 serving as a mask for dry etching is formed on both surfaces by thermal oxidation.

  (B) A resist pattern is formed on the silicon oxide film 32 by photolithography using a double-sided aligner, and the silicon oxide film 32 is patterned by oxide film etching using hydrofluoric acid using the resist pattern as a mask. As a result, an oxide film pattern 32a (see FIG. (B-1) for a plan view) that determines the shape of the reagent inlet and the buffer inlet is formed on the upper surface of the silicon substrate 30, and the reaction chamber shape is formed on the lower surface of the silicon substrate 30. An oxide film pattern 32b (see FIG. 2B-2 for a plan view) is formed.

  (C) The silicon substrate 30 is dry-etched using ICP (Inductively Coupled Plasma) -RIE using the oxide film pattern 32a of the silicon substrate 30 on the upper surface side and the lower surface side as a mask. The reaction chamber 2 is formed on the lower surface side of the substrate with a through hole serving as the buffer inlet 12.

(D) The silicon oxide films 32 on both sides of the silicon substrate 30 are removed.
(E) A bottom plate 6b having a mesh structure and a flat glass substrate 36a are prepared. As the bottom plate 6b, for example, a glass substrate having a mesh structure in which a large number of pores are formed by dry etching on a glass substrate having a thickness of about 100 μm can be used. In the glass substrate 36a, a circular recess serving as a bottom is formed by dry etching, and a groove serving as the liquid discharge port 14 is formed around the recess. The bottom surface of the recess becomes the bottom surface 6a of the reaction chamber.
Then, the bottom plate 6b is bonded to the lower surface of the silicon substrate 30 by the method described above. Thereafter, the glass substrate 36a is bonded onto the bottom plate 6b so that the concave portion is on the inside. The bottom plate 6b and the glass substrate 36a can be joined using, for example, hydrofluoric acid.

The dimensions and materials shown in the manufacturing method are examples, and the present invention is not limited to these.
In the above-described embodiments, cells are the target of analysis, but the application is not limited to cell analysis, but can be applied if the purpose is to allow a fluid reagent to react only at a specific site. It is.

  INDUSTRIAL APPLICABILITY The present invention can be used as a minute reaction apparatus that causes a reaction by selectively reacting a part of various solid samples including cells, and can be used as, for example, a cell culture apparatus. .

It is a figure which shows roughly the structure of the micro reaction apparatus of this invention, (A) is a perspective view, (B) is sectional drawing in the (A) AA line position. It is a figure which shows the micro reaction apparatus of one Example, (A) is a schematic perspective view, (B) is a top view, (C) is a bottom view. It is a figure which shows the computer analysis of the mode of reagent introduction | transduction in the Example, (A) is a perspective view which shows the external shape of a reaction chamber, (B) is a reagent concentration distribution in the bottom face of a reaction chamber, (C) is a reaction chamber. The reagent concentration distribution in the cross section is shown. It is sectional drawing which shows the observation method by the fluorescence microscope and the confocal fluorescence microscope in the Example. It is an image which shows the result of having observed the flow of the reagent on a bottom surface with a fluorescence microscope. It is an image which shows the result of having observed the flow of the reagent of the depth direction with the confocal fluorescence microscope. It is a figure which shows the micro reaction apparatus of another Example, (A) is a schematic perspective view, (B) is sectional drawing. FIG. 3 is a process end view showing a method for manufacturing the microreaction apparatus of the embodiment of FIG. 2. FIG. 8 is a process end view showing a method for manufacturing the microreaction apparatus of the embodiment of FIG. 7.

Explanation of symbols

2 Reaction chamber 4 Upper surface 6 Bottom 6a Bottom surface 6b Bottom plate 8 Sample 10 Reagent introduction port 10a Reagent flow 12 Buffer introduction port 12a Buffer flow 14 Liquid discharge port

Claims (9)

In a microreaction apparatus that is formed in a cylindrical shape having an upper surface and a bottom surface, and that has a reaction chamber for containing a solid sample at the bottom inside, in a substrate,
The reaction chamber is
At least one reagent introduction port disposed on the upper surface and introducing a reagent into the reaction chamber;
A three or more buffers inlet for introducing a buffer fluid into the reaction chamber, the buffer inlet the buffer inlet port is disposed so as to surround the reagent inlet in said top surface,
A plurality of liquid outlets arranged at the bottom corner to discharge the liquid in the reaction chamber to the outside ;
A microreaction apparatus comprising: The microreaction apparatus according to claim 1 , wherein the buffer inlets are arranged at equal intervals on one circumference centered on the reagent inlet. The microreaction apparatus according to claim 1 or 2 , wherein three or more liquid discharge ports are provided. The microreaction apparatus according to claim 3 , wherein the liquid discharge ports are arranged at equal intervals in the bottom corner. The microreaction apparatus according to any one of claims 1 to 4 , wherein the reaction chamber is cylindrical. The bottom of the reaction chamber has a double structure consisting of a bottom surface and a bottom plate that is spaced from the bottom surface and holds the sample,
The bottom plate has a plurality of holes smaller than the sample to be held, a liquid discharge port is provided below the bottom plate, and the reagent and buffer fluid introduced into the reaction chamber pass through the holes in the bottom plate. The microreaction apparatus according to any one of claims 1 to 5 , wherein the microreaction apparatus is discharged from a liquid discharge port. A plurality of samples are held at the bottom of the reaction chamber, and the flow direction of the reagent introduced from the reagent inlet is controlled by adjusting the flow rate of the buffer fluid introduced from each of the buffer inlets. The microreaction apparatus according to any one of claims 1 to 6 , wherein the microreaction apparatus is selectively acted on a part of a sample. The microreaction apparatus according to any one of claims 1 to 7 , wherein the sample held in the reaction chamber is a living cell. The microreaction apparatus according to any one of claims 1 to 8 , further comprising a buffer fluid introduction mechanism connected to each of the buffer introduction ports and capable of adjusting a flow rate of the introduced buffer fluid.