JP3654481B2 - Microreactor for biochemical reaction - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a biochemical reaction microreactor for simultaneously performing a large number of biochemical reactions in parallel.
[0002]
[Prior art]
In biochemical experiments, generally, complicated operations such as fractionation, mixing, reaction, detection, separation and the like are required for reagents in the order of μL. In particular, when dealing with unstable substances such as proteins and RNA, consideration of the experimental environment, such as measures against temperature and degrading enzymes, is indispensable. It has a considerable impact on
[0003]
Conventionally, in such biochemical experiments, a method has been adopted in which different reagents are put in a large number (for example, 48) of reaction chambers, and the whole is simultaneously processed under the same conditions to detect the optimum conditions. However, if this method is applied as it is to a larger number of reaction tests (for example, 1000 or more), the test apparatus becomes very large, which makes it impossible to process and inspect the whole under the same conditions. .
[0004]
On the other hand, the use of microscale channels of 1 mm or less, such as capillary electrophoresis, has made it possible to reduce the size of the apparatus and increase the analysis speed (Proc. Of HPCE '93, Orlando, 1993). In recent years, research on the application of so-called MEMS (Microelectromechanical Systems) Technology (Proc. Of MEMS '97, Nagoya, 1997) to chemical analysis and genetic analysis, for example, in the United States, is related to technical issues related to the Human Genome Project (“Microfabrication Technology for Biomedical Applications ", Cambrige Health Institute, 1996), and μTAS (" Micro Total Analysis System ", Kluwer Academic, 1995) are discussed in Europe.
[0005]
[Problems to be solved by the invention]
The specific systems realized in these studies are mainly for the purpose of analysis targeting DNA molecule handling and quantification, in which technical issues associated with system fabrication and integration are the main research subjects. ing. Therefore, the conventional system described above cannot cope with a wide range of biochemical experiments including substance synthesis reactions such as protein synthesis.
[0006]
Further, US Pat. No. 5,589,136 (“SILICON-BASED SLEEVE DEVICES FOR CHEMICAL REACTIONS”) includes a sleeve reaction chamber and heating means for the sleeve reaction chamber, as schematically shown in FIG. A microchemical reactor is disclosed wherein the sleeve reaction chamber has a groove into which an insert or liner can be inserted. In this figure, 1 is a microchemical reactor, 1a is recessed, 2 is a power supply / control system, 3 is a syringe, 4 is a window of silicon rubber, 5 is an electromagnetic coil, 6 and 7 are electrodes, C is a capacitor, and L is It is a coil.
[0007]
However, although this microchemical reactor can perform DNA reaction, DNA growth, DNA synthesis, etc., it is difficult to integrate a large number of reactors (for example, 1000 or more). There was a problem that was difficult to do.
[0008]
The present invention has been developed to solve the above-described problems. That is, the main object of the present invention is to provide a bioreactor microreactor capable of simultaneously performing, for example, a large number of biochemical reactions of 1000 or more in parallel. Another object of the present invention is to provide a bioreactor microreactor capable of performing not only a simple analysis but also a substance synthesis reaction such as protein synthesis on a cell.
[0009]
[Means for Solving the Problems]
According to the present invention, the reaction chamber includes a plurality of independent reaction chambers formed on the surface of the silicon substrate by anisotropic etching, and a flat plate that is anodically bonded to the surface of the silicon substrate and seals the reaction chamber. Is formed to be small enough to be integrated on a single silicon substrate having a diameter of 30 cm, and has an injection port and an exhaust port, and a channel communicating the injection port and the exhaust port. The biochemical reaction characterized in that the port and the discharge port have a through-hole communicating with the back surface of the silicon substrate, supply the reagent to the injection port through the through-hole, and discharge from the discharge port. A microreactor is provided.
[0010]
According to the configuration of the present invention described above, since there are a plurality of independent reaction chambers sealed with a flat plate on a silicon substrate, a plurality of biochemical reactions can be simultaneously performed in parallel in each reaction chamber. In particular, since this reaction chamber is formed on the surface of a silicon substrate by anisotropic etching, for example, if one reaction chamber is constituted by 4 mm × 10 mm, 1000 or more reactions are performed on a single silicon wafer having a diameter of 30 cm. A chamber can be formed. Therefore, by simultaneously performing 1000 or more biochemical reactions in parallel in this single silicon wafer, the whole can be processed and inspected under the same conditions, and the optimum conditions can be easily determined. In addition, each reaction chamber can be sized to match the size of an animal cell, making it possible to create an environment close to the inside of the cell. In addition to simple analysis, substance synthesis reactions such as protein synthesis can be performed on the cell. Can do.
[0011]
The reaction chamber includes an injection port and a discharge port, and a channel that communicates the injection port and the discharge port. The injection port and the discharge port include a through hole that communicates with the back surface of the silicon substrate. Since the reagent is supplied to the injection port through and discharged from the discharge port , different reagents can be continuously supplied to the injection port, and different reactions can be performed simultaneously in parallel for a long time.
[0012]
Further, at least a part of the flat plate is a transparent portion, and an internal reaction is optically observed through the transparent portion. With this configuration, reactions in a number of reaction chambers can be observed simultaneously using a high-sensitivity film, a CCD camera, or the like, and an optimal cell can be easily detected by image processing or the like.
[0013]
Furthermore, it is preferable that a measurement circuit is formed on a part of the silicon substrate. With this configuration, various sensors, temperature controllers, oscillators, etc. can be incorporated using the same circuit manufacturing technology as integrated circuits, and many biochemical reactions can be performed simultaneously in parallel and efficiently controlled and data Can be obtained.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. In each figure, common portions are denoted by the same reference numerals, and redundant description is omitted.
The present invention aims to develop a system that enables large-scale systematic experiments by realizing the operations performed on the above-described general workbench on a micro system, and automating and speeding up the operation. It is. Hereinafter, this system is named “Micro Work Bench” (MWB) in the sense that the work bench is micro-sized.
[0015]
As a merit of micro-system, in general,
(1) The dead volume is small and analysis is possible at high speed with a small amount of sample.
(2) A small and lightweight experimental system can be realized.
(3) Easy integration and parallelization.
(4) Mixing of impurities can be suppressed.
(5) Operation errors can be reduced by systematization.
A wide range of applications can be expected.
[0016]
FIG. 1 is a diagram schematically showing the concept of the microreactor of the present invention. This microreactor realizes a series of biochemical experimental operations by mixing a solution, performing a reaction, quantifying and analyzing and then separating them by a combination of several cells.
In the figure, for example, (1) a reservoir cell, (2) a mixing cell, (3) a reaction cell, (4) a detection cell, and (5) a separation cell An example of a combination of (Separation Cell) is shown.
[0017]
Furthermore, if these cells are further integrated by taking advantage of microfabrication, a system capable of performing multi-stage reaction separation operations in parallel can be considered. For example, molecular evolution and combinatorial chemistry ( A system that can handle a wide range of biochemical experiments such as Combination Chemistry).
[0018]
FIG. 2 is a conceptual diagram similar to FIG. As shown in this figure, if one reaction chamber 11 is constituted by 4 mm × 10 mm, for example, 1000 or more reaction chambers can be formed on a single silicon wafer having a diameter of 30 cm. Therefore, by simultaneously performing 1000 or more biochemical reactions in parallel in this single silicon wafer, the whole can be processed and inspected under the same conditions, and the optimum conditions can be easily determined. This is only an example, and by making the reaction chamber 11 smaller, a larger amount of reactions can be performed simultaneously.
[0019]
【Example】
(Example 1)
3A and 3B are diagrams showing an embodiment of the microreactor of the present invention, in which FIG. 3A is a plan view and FIG. 3B is an enlarged cross-sectional view taken along line AA.
In order to realize the system as described above, in addition to elemental technologies related to liquid feeding, reaction, detection, separation, etc., there are various problems such as connection problems between cells including flow paths and control problems as a whole system. It is necessary to solve various technical problems. As a first step for examining these problems, a microreactor for mixing two liquids as shown in FIG.
[0020]
In FIG. 3, a channel 13 having a depth of h = 20 μm is dug on a 20 mm × 20 mm silicon substrate 12 by anisotropic etching. Each sample has two injection ports 14 (Inlet) and one discharge port 15 (Outlet). Provided. Two types of T-shaped and h-shaped channel shapes, and three types of 200 μm, 400 μm, and 800 μm channel width w were manufactured, for a total of six types of reactors.
[0021]
FIG. 4 is a diagram showing the manufacturing method of FIG. As shown in this figure, (A) First, the channel 13 is processed on the silicon substrate 12 by anisotropic etching. Anisotropic etching is one of micromachining techniques, and is a technique for obtaining a high aspect ratio structure with less undercut in wet etching. This can be achieved, for example, by crystal orientation dependent etching using KOH or ethylenediamine aqueous solution as an etchant.
[0022]
(B) Next, a through hole 16 communicating with the back surface is provided in the injection port 14 and the discharge port 15. This processing can also be performed by anisotropic etching. (C) Next, the flat plate 17 is anodically bonded to the surface of the silicon substrate 12. The flat plate 17 is preferably at least partially transparent. By providing such a transparent part, an internal reaction can be optically observed through the transparent part.
[0023]
For the flat plate 17, for example, heat resistant glass (trade name: Pyrex) or the like is preferably used. Further, anodic bonding is usually performed by applying a voltage between the two while being heated to about 300 to 500 ° C. By this anodic bonding, the channel 13 closed by the flat plate 17 functions as the reaction chamber 11. (D) Finally, the eyelet 18 is bonded to the back surface of the silicon substrate 12 using an adhesive (for example, epoxy).
[0024]
As described above, the biochemical reaction microreactor of the present invention includes a plurality of independent reaction chambers 11 formed on the surface of the silicon substrate 12 by anisotropic etching, and the reaction chamber 11 that is anodically bonded to the surface of the silicon substrate 12. And a flat plate 17 that seals.
The reaction chamber 11 also has an injection port 14 and an exhaust port 15 and a channel 13 that communicates the injection port and the exhaust port. The injection port 14 and the exhaust port 15 communicate with the back surface of the silicon substrate. The reagent is supplied to the injection port 14 through the through-hole 16 and discharged from the discharge port 15. Further, at least a part of the flat plate 17 is a transparent portion, and an internal reaction can be optically observed through the transparent portion. Further, if necessary, a measurement circuit (various sensors, temperature controller, oscillator, etc.) is formed on a part of the silicon substrate 12.
[0025]
(First Experiment Example)
FIG. 5 is a first experimental example using the microreactor of FIG. In the experiment, a silicon tube 19 is connected to the injection port 14 and the discharge port 15 as shown in this figure, and a sample is injected from the microsyringe 21 using a syringe pump or the like. Two types of samples come into contact with each other at the junction, and the channel portion downstream from the junction serves as a reactor to allow the samples to react with each other. In addition, when injecting a sample with the syringe 21, in order to avoid that a flow is blocked by air bubbles etc., it is necessary to perform what is called priming to fill the inside of the reactor with a liquid.
[0026]
In order to conduct basic studies on biochemical reactions in microsystems, specific reaction experiments were conducted in the fabricated microreactor. In the microreactor shown in FIG. 3, the injection port 14 includes a first port 14a for a mixed solution of luciferase and luciferin and a second port 14b for an ATP solution, and the channel 13 includes a first port 14a and a second port. 14 b, and a reaction channel 13 b that communicates the merge channel 13 a and the discharge port 15. In addition, the firefly luciferase reaction in the reaction channel 13b is optically observed through the transparent portion.
[0027]
That is, in the present invention, the luminescence reaction by firefly luciferase is taken up as a biochemical reaction that is relatively easy to detect. The firefly luciferase reaction is a luminescent reaction in which an enzyme called luciferase proceeds using luciferin and ATP (adenosine triphosphate) as substrates, and light is emitted with the oxidation of luciferin, so the luminescence intensity at the channel portion should be monitored. To know the activity of the reaction.
[0028]
In the experiment, luciferin used for ATP detection, a detection reagent containing luciferase (Sigma # L0633) and an ATP aqueous solution were injected from the injection port, and then the reactor was photographed using a high-sensitivity film to observe luminescence. went.
[0029]
FIG. 6 is a diagram showing the test results of FIG. As a result of photographing, as shown in FIG. 6, it was confirmed that the detection reagent and ATP were gradually mixed in the reactor and emitted light. There were no problems such as clogging of the protein solution in the mechanical part such as liquid feeding in the microchannel, and although it had a simple structure, many basic findings were obtained toward the realization of the microreactor.
[0030]
(Second Experimental Example)
FIG. 7 shows a second embodiment using the microreactor of FIG. In order to evaluate the possibility of protein synthesis, two mixed solutions A and B that are known to synthesize proteins when mixed are prepared, and the mixture A contains messenger RNA (mRNA) None were tested. That is, as the mixed solution A, a mixed solution of L- [ ¹⁴ C] -phenylalanine, ATP, GTP, mRNA, phosphoenolpyruvate, pyruvate kinase, polyuridylic acid is used, and as the mixed solution B, ribosome, soluble protein factor A mixture of tRNA was used.
[0031]
As shown in FIG. 7, the injection port 14 of the microreactor used in this experiment is composed of a first port 14a for the mixture A and a second port 14b for the mixture B, and the channel 13 is the first port 14a. And the second channel 14 b and the reaction channel 13 b that communicates the merge channel 13 a and the discharge port 15. In addition, the silicon substrate 12 was floated in warm water maintained at 37 ° C., and the temperatures of the reaction chamber 11 (that is, the reaction channel 13b) and the discharge port 15 were measured with a thermocouple. During the reaction, all equipment except the microreactor was kept at a low temperature of 2 ° C. to 4 ° C. to prevent reaction outside the microreactor.
[0032]
The reaction was triggered by mixing equal amounts of the two mixtures A and B. This equal volume mixing was performed for 100 minutes using a micro syringe 21 at a constant flow rate of 0.05 μL / min. The mixed reaction solution discharged from the discharge port 15 of the microreactor is collected on a filter paper 22 (Whatmann 3 mm), boiled for 20 minutes with 10% TCA solution (trichloroacetic acid), and further twice at room temperature for 10 minutes with the same solution. It was washed and further washed with ethanol twice for 10 minutes at room temperature. After drying, the radioactivity of insoluble TCA remaining on the filter paper 22 was measured. For the measurement, the mixture A containing polyuridylic acid as messenger RNA and the one not containing polyuridylic acid were tested under the same conditions.
[0033]
FIG. 8 is a diagram showing the test results of FIG. In this figure, the horizontal axis is the detected amount of ¹⁴ C, and (a) on the vertical axis is the case where mRNA is not included, and (b) is the complete case including this. In the complete system of (b), about twice as much ¹⁴ C is detected as compared with the case where the mRNA of (b) is not included. Therefore, it can be seen that the uptake of ¹⁴ C is enhanced by mRNA, and that a protein (polyphenylalanine) corresponding to the genetic code on mRNA was successfully synthesized in the microreactor.
[0034]
In addition, this invention is not limited to the Example mentioned above, Of course, it can change variously in the range which does not deviate from the summary of this invention.
[0035]
【The invention's effect】
As described above, the bioreactor microreactor according to the present invention can simultaneously perform a large number of biochemical reactions of, for example, 1000 or more in parallel, and can perform not only simple analysis but also substance synthesis reactions such as protein synthesis. Can also be performed on the cell.
[Brief description of the drawings]
FIG. 1 is a diagram schematically showing the concept of a microreactor of the present invention.
FIG. 2 is a conceptual diagram similar to FIG.
FIG. 3 is a diagram showing an embodiment of a microreactor of the present invention.
4 is a diagram showing the manufacturing method of FIG. 3; FIG.
FIG. 5 is a first experimental example using the microreactor of FIG. 3;
6 is a halftone image displayed on the display showing the test results of FIG. 5. FIG.
FIG. 7 is a second experimental example using the microreactor of FIG. 3;
FIG. 8 is a diagram showing the test results of FIG.
FIG. 9 is a configuration diagram of a conventional microreactor.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Micro chemical reactor 2 Power supply / control system 3 Syringe 4 Silicom rubber window 5 Electromagnetic coils 6, 7 Electrode 11 Reaction chamber 12 Silicon substrate 13 Channel 13a Merge channel 13b Reaction channel 14 Injection port 14a First port 14b Second port 15 Discharge port 16 Through hole 17 Flat plate (heat-resistant glass)
18 Eyelet 19 Silicon tube 21 Micro syringe 22 Filter paper

Claims (5)

A plurality of independent reaction chambers formed by anisotropic etching on the surface of the silicon substrate, and a flat plate which is anodically bonded to the surface of the silicon substrate and seals the reaction chamber ,
The reaction chamber is formed to be small enough to be able to be integrated on a single silicon substrate having a diameter of 30 cm, and has an injection port and a discharge port, and a channel communicating the injection port and the discharge port. The injection port and the discharge port have a through-hole communicating with the back surface of the silicon substrate, supply the reagent to the injection port through the through-hole, and discharge from the discharge port.
A microreactor for biochemical reaction characterized by the above.   The bioreactor microreactor according to claim 1, wherein at least a part of the flat plate is a transparent part, and an internal reaction is optically observed through the transparent part.   The bioreactor microreactor according to claim 1, wherein a measurement circuit is formed on a part of the silicon substrate. The injection port includes a first port for a luminescent enzyme and a second port for a substrate solution. The channel includes a merging channel that communicates the first port and the second port, and the merging channel and a discharge port. The bioreactor microreactor according to claim 2 , characterized in that the bioluminescence reaction in the reaction channel is optically observed through the transparent portion. The injection port includes a first port for a mixed solution A containing a nucleic acid having genetic information and a second port for a mixed solution B, and the channel is a merging channel that communicates the first port and the second port. And a reaction channel that communicates the merging channel and the discharge port, the silicon substrate is held at a predetermined temperature from the outside, and thereby a protein synthesis reaction according to the genetic code on the nucleic acid occurs in the reaction channel. The bioreactor microreactor according to claim 2 .

Images