JP2009022954A - Extraction system, extraction method and system and method for synthesizing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an extraction system using a microreactor capable of easily manufacturing a stable molecular film and an analysis system, and to provide an extraction method and a system and a method for synthesizing protein. <P>SOLUTION: The extraction system is provided with: a forward extraction part 91 in which a substance such as a biologically-relevant substance is extracted forwardly in a reversed micelle solution and the refolding of the denatured protein is performed in a microextraction system (a reversed micelle type microreactor); a raw material recovery part 92 for recovering a raw material aqueous solution; a back extraction part 93 for subjecting the substance such as the biologically-relevant substance extracted by the forward extraction to back extraction; a circulation part 95 for circulating the reversed micelle solution; and a product recovery part 94 for recovering extraction products obtained by the back extraction. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an extraction system, an extraction method, a protein synthesis system, and a protein synthesis method.

(1) Background Art Regarding Application of Microreactor and Method for Fabricating Lipid Bilayer Membrane In conventional large reaction systems, various reaction engineering problems arise due to scale-up of the reaction system. These problems can be cited as follows, for example. The homogenization in the system by stirring and mixing tends to cause concentration and temperature distribution. The larger the reaction system, the more likely it is that the heterogeneity will occur. In a tube-type reaction, when the heat of reaction in the tube is high, a temperature distribution is generated in the tube, so that the reaction rate cannot be controlled, and further, a by-product is easily formed, and a large amount of by-product causes a great load on the environment. Moreover, in order to maintain the uniformity of the temperature and concentration of the system, the agitation power becomes large, so it is not an energy saving type.

  As a system that can solve the above problems, a microreactor system can be considered. The main reaction engineering features of the microreactor are as follows.

1) Almost no side reaction occurs.

2) The reaction temperature can be easily controlled and the reaction rate can be controlled.

3) Since the concentration distribution hardly occurs, it is easy to scale up the reaction system.

4) Since the specific surface area and specific interface area are large, the molecular diffusion distance is short, and the diffusion-controlled reaction is dramatically accelerated.

5) Since an agitation power for causing forced convection is not required, an energy-saving reaction system can be constructed.

6) Since the reaction heat can be quickly diffused, a rapid reaction can be caused.

  As mentioned above, the microreactor has the characteristics of high efficiency, energy saving, low environmental load and rapid reaction, which enables the reaction of ultra-trace components contained in ultra-trace reagents related to environment, medicine, pharmaceutical, food industry and science. It seems to be very useful as a place. Furthermore, since integration of microreactors enables realization of mass production of useful substances, it has recently received much attention as an industrial production method for new useful substances, and research on these has been actively conducted.

  On the other hand, in order to use a microreactor as a field for chemical reaction, researches such as enzyme immobilization in a microchannel are being conducted. For example, production of a polymer film, enzyme immobilization on the polymer film, and enzyme reaction have been attempted by interfacial polymerization at the water / oil interface in the microchannel (for example, see Non-Patent Document 1). However, in this method, it is difficult to immobilize various enzymes, and it is difficult to regenerate a deteriorated film. Further, since it is a polymer film, there is a problem that the film does not flow.

  In addition, the production of useful substances using conventional biopolymers has the following problems.

1) Low productivity of the target substance.

2) Accumulation of products in cells.

3) Formation of inert, insoluble aggregates.

4) There are various problems that reduce the recovery efficiency and selectivity of the target substance.

  On the other hand, research and development on a method for forming a lipid bilayer membrane that plays an active role as a model of a biological membrane has been actively conducted. Already, patch-clamp (see Non-Patent Document 2, for example), brush technique, dipping technique, bubble technique, syringe technique, simple technique A method for producing a monolayer technique has been developed (see, for example, Non-Patent Document 2). In addition, a method for producing a lipid bilayer is being developed by a method such as an automatic lipid droplet discharge method (for example, see Patent Document 1). However, all of these methods have the following problems.

1) It takes time to prepare a lipid bilayer by natural thinning of the membrane.

2) The area of the film is small, and its application is limited.

3) It is difficult to scale down or scale up the system.

4) The membrane is unstable.

5) Since there is no fluidity of the membrane, it is difficult to renew and continuously use the membrane.

Although there are problems as described above, molecular membranes such as lipid bilayer membranes, reverse bilayer membranes, monomolecules, micelles, reverse micelles, etc., are produced by various methods, and application to various fields, particularly sensor fields, is attempted. It has been. In addition, research on the preparation of polymer membranes in the microreactor and enzyme immobilization on the polymer membranes has been carried out. For example, the formation of molecular membranes in the microchannel, such as the liquid / liquid laminar interface and the liquid / There are no precedents created between gas-laminous interfaces and the like. Moreover, the present condition is that the rapid and large area preparation technique of the lipid bilayer membrane and microdomain-like functional membrane which are biological membranes is also not established.
H. Hisamoto, et al., Anal. Chem., 2003, 75, 350-354. Neher E., Sakmann B. “Single Channel Recording”, Plenum Press, New York (1983) Tien HT, “Bilayer Lipid Membranes: Theory and Practice”, Marcel Dekker, Inc., NY, pp.672 (1974) ) Japanese Patent Application No. 2001-91494 (paragraph “0008”, FIG. 1) (2) Background Technology for Detecting Amyloid-type Proteins and Viruses Conventionally, base sequence information relating to various substances is grasped by remarkable progress in genome structure analysis. It became possible. However, the structural analysis of the genome can be elucidated with the static putative structure of the gene candidate and its transcription product (protein). It is extremely difficult to grasp dynamic information such as protein conformational changes accompanying changes in external environmental conditions. In other words, the DNA chip targets samples having the same base sequence, the same base sequence, that is, the amino acid sequence (primary structure) of the protein biosynthesized therefrom is the same, and the higher order structure (secondary structure, tertiary structure). At present, it is impossible to detect different structures or quaternary structures.

  On the other hand, the function of a protein is closely related to its higher-order structure, and the structural change of the protein due to external environmental conditions impairs the original biological function. For example, amyloid fibril formation and amyloid fibril deposition due to protein structural abnormalities can cause mad cow disease, human Creutzfeldt-Jakob disease, Kuru disease, sheep scrapie and other prion diseases, Alzheimer's dementia, dialysis amyloid-cis It is known to cause diseases such as medullary thyroid cancer, AL amyloidosis, familial amyloid polyneuropathy, familial amyloidosis, Parkinson's disease, and Huntington's disease.

  Amyloid fibers are formed from amyloid-type proteins or peptides of about 10 residues, and the disease caused by the deposition of amyloid fibers is called amyloid disease (or amyloid-cis).

  Prion disease, a type of amyloid disease, is a fatal neurodegenerative disease, and no cure has yet been found (Nature, vol422, 2002). Alzheimer's has nearly 4 million people in the United States alone, and is expected to triple that in the next 30 years (Nature Medicine, 4, 2003.).

  Dialysis amyloidosis is one of the “iatrogenic diseases” (diseases created by medical practice) seen in patients who have been on blood dialysis for more than 10 years. Currently, about 200,000 people each in the US and Japan are receiving hemodialysis treatment. Although this symptom manifests as wrist pain and movement disorder called carpal tunnel syndrome, an effective treatment has not yet been established.

  On the other hand, viruses such as human immunodeficiency virus (HIV) contain a nucleic acid and a transcriptase that are viral genetic information and are encapsulated in a lipid membrane. By fusion of the viral membrane and cell membrane, the viral nucleic acid and the transcriptase become intracellular in the host cell. To be released. In the host cell, DNA is synthesized by HIV reverse transcriptase to produce new HIV in the body. This HIV specifically binds to important lymphocytes (T cells) that control immunity, making a large number of copies in the lymphocytes and destroying the lymphocytes, eventually humans become immune deficient, To death.

  Accordingly, early diagnosis of various diseases and HIV associated with structural abnormality of the protein and HIV is indispensable and considered to be extremely important. It is also necessary to elucidate the mechanisms of various diseases and accelerate the development of new drugs.

  On the other hand, as a detection method for a biological sample, there are conventionally various detection methods based on interactions between biomolecules. For example, there are a bioassay and a method for detecting a target substance by labeling an antigen or antibody with an enzyme (ELISA method), a quartz crystal resonator method, a gel shift assay method, a surface plasma resonance (SPR) method, a microcalorimetry method and the like. The detection range of these detection methods is about 10 ng to 100 pg. Among these, the measurement time of the ELISA method and the gel shift assay method is very long, and it is not suitable for the measurement of change with time like rapid measurement, high sensitivity measurement, and microcalorimetry. The surface plasma resonance (SPR) method makes it difficult to detect the absolute amount of the sample.

  In addition, in recent years, a quartz crystal resonator that is expected as a sensor can detect a biological sample of ˜10 ng, but in order to have a function as a sensor, a series of complicated reactions are performed. Various sample recognition modifying groups must be modified on the electrode surface. However, detection of samples by surface modification is an irreversible reaction, and it is impossible to repeatedly use electrodes, and there are still many unsolved problems in terms of speed, simplicity, economy, and versatility. Yes.

  As described above, conventional measurement methods for biological samples also have various problems, and in particular, ultra-trace, rapid, high sensitivity, simple, economical to distinguish structural abnormalities of proteins such as amyloid type proteins. No effective detection method has been developed yet.

(3) Background technology related to bio-related substance extraction using reverse micelles Bioproducts range from low-molecular substances such as amino acids, sucrose and ethanol to high-molecular substances such as proteins, starches and polynucleotides (nucleic acids). There are a wide variety of substances. Many of these substances lose their original physiological functions by being easily denatured and deactivated by heat, pressure, acid or alkali. Furthermore, since these usually exist as a dilute multi-component mixed solution, it is not easy to separate and purify the target substance stably and highly selectively. Thus, it is very important to separate and purify useful biological substances from bioproducts in a manufacturing process for pharmaceuticals, foods, chemical products, etc., quickly, efficiently and selectively.

  Conventional reverse micelle extraction includes an injection method, a liquid phase contact method and the like, but these systems usually have the following problems when extraction is performed using a large reaction tank.

  1) The extraction speed is slow.

  2) Degeneration / inactivation of biological materials is likely to occur.

  3) It is difficult to control the temperature of the system.

  4) The larger the extraction system, the more likely it becomes non-uniform.

  That is, in order to increase the extraction speed, it is necessary to increase the oil-water interface area, and thus stirring must be performed. When the stirring power is large, the dispersion droplet diameter of the emulsion is reduced, and the oil-water interface area is increased. This increases the mass transfer rate at the oil / water interface and increases the extraction rate. However, since a surfactant is present in the system, emulsification proceeds by stirring and phase separation becomes difficult. For this reason, reverse micelle extraction is variable in scale, but at present it is difficult to perform extraction on a large scale. In addition, there is a problem that stirring causes protein shearing denaturation and denaturation / inactivation.

  The larger the reverse micelle extraction system is, the more difficult it is to reproduce a stable reverse micelle structure. Furthermore, since surfactants easily take various molecular aggregate forms such as reverse vesicles, it is difficult to control the size of reverse micelles.

  In addition to this, there is a hollow fiber module method, but in this method, it is difficult to adjust the liquid feeding pressure to each phase through a membrane for forming a stable reverse micelle. Therefore, its practical use has not been realized yet.

  Furthermore, aggregated proteins produced as inclusion bodies in cells are denatured using genetic recombination techniques. Therefore, the current situation is that cells must be first destroyed, a denaturant is added to dissolve the protein, and then refolding must be performed to restore the collapsed protein's three-dimensional structure.

(4) Background art of cell-free protein synthesis Living cells biosynthesize target proteins from messenger RNA (mRNA) encoding proteins by the action of transfer RNA, proteinaceous translation factors, and the like. Similar protein synthesis can be performed in vitro using an extract derived from living cells or a cell disruption solution. Such a synthesis system is called cell-free protein synthesis. In this system, transcription and translation may be coupled using DNA as a template. Furthermore, since there is no restriction of life maintenance of living cells, the degree of freedom of the system is dramatically increased. Therefore, it has the following characteristics.

  1) Unnatural amino acids and radiolabeled amino acids can be freely used as constituents in proteins.

  2) Proteins that are toxic in cells can also be synthesized in this system.

  3) Proteins can be synthesized quickly and simultaneously from multiple templates.

  4) There are no restrictions on the type and concentration of substances added to this system.

  5) The time required for the synthesis reaction is generally short.

  However, since the cell fluid used in conventional cell-free protein synthesis systems is extracted by cell crushing, the concentration of the cell fluid is low and the extraction conditions are harsh. Inactivation of ribosome, which is one of the translation factor groups, occurs. Therefore, when synthesizing proteins in an actual cell-free system, the duration of the synthesis reaction is short, and the yield obtained is only about 0.1% to 1% of living cells, which is not practical as a protein synthesis method. Yes.

  (1) Therefore, in view of the above-described problems in the prior art, the present invention includes a monolayer, a bilayer, a reverse bilayer, a lipid bilayer, a molecular multilayer, a reverse micelle, and a micelle in a microreactor. Providing automated, rapid, and continuous production methods of molecular membranes, as well as various rapid chemical reactions, bioreactors, structurally and denatured protein refolding, separation (extraction) systems and analysis systems using this system It is for the purpose.

  (2) In addition, the present invention provides a trace amount of these substances and viruses in a simple manner by interacting with a molecular film that is automatically, rapidly and continuously produced in a structurally abnormal protein (amyloid type protein) or virus microreactor. Providing rapid and sensitive detection microchips, biosensing systems, or providing early diagnosis and treatment systems for intractable diseases by refolding and separating (extracting) structurally abnormal proteins using microreactors equipped with molecular membranes It is intended to do.

  (3) In addition, the present invention is simple, short-time, energy-saving, and mild reaction / extraction conditions, high-efficiency and rapid extraction of substances, high activity, high-efficiency and rapid extraction of biological materials such as proteins, denatured proteins It is an object of the present invention to provide a micro-extraction system using reverse micelles that performs rapid refolding.

  (4) Another object of the present invention is to provide a biomimetic tampane synthesis system using a microreactor equipped with a molecular film and a microextraction system using reverse micelles.

  In order to achieve the above object, the first feature of the present invention is that a microchannel, a first inlet for introducing a first fluid into the microchannel, and a second for introducing a second fluid into the microchannel. An inlet, and a third inlet provided between the first inlet and the second inlet for introducing the amphiphilic molecule mixture containing the amphiphilic molecule into the microchannel; The gist is that the microreactor forms a molecular film on the boundary surface between the first fluid and the second fluid.

  The second feature of the present invention is that a microchannel, a first inlet for introducing a first fluid into the microchannel, and an amphiphilic molecule mixture containing amphipathic molecules are introduced into the microchannel. And a microreactor that forms a molecular film on the boundary surface between the first fluid and the wall surface of the microchannel.

  According to the microreactor according to the first and second features, automatic, rapid and continuous production of molecular membranes such as monolayer, bilayer, reverse bilayer, lipid bilayer, molecular multilayer, reverse micelle and micelle It can be performed.

  In addition, the molecular film generated in the microreactor according to the first and second features is a monomolecular film, a bimolecular film, a lipid bimolecular film, an inverted bimolecular film, a molecular multilayer film, a micelle, an inverted micelle, or a vesicle. , A reverse vesicle, an emulsion, an LB film, or a structure in which these are mixed.

  Further, the molecular film generated in the microreactor according to the first and second features may have a shape in which a planar structure and a three-dimensional structure are mixed.

  In addition, the amphiphilic molecule mixture in the microreactor according to the first and second features includes proteins, nucleic acids, glycolipids, cholesterol, fluorescent dyes, ligands, photosensitive molecules, cross-linking agents, ion channels, and electron conjugate systems. It is desirable to include one or more substances, polymerizing agents, co-surfactants, crown ethers, fullerenes, carbon nanotubes, porphyrins, molecular tongs, redox agents, steroids, cyclodextrins, polypeptides, and peptides.

  Further, the microchannel of the microreactor according to the first and second features may be any one of a straight tube, an elbow, a bend, or a structure in which these are mixed.

  The wall surface of the microchannel of the microreactor according to the first and second features may be any one of a smooth shape, an uneven shape, a surface modification, or a structure in which these are mixed.

  Moreover, you may integrate the microchannel of the microreactor which concerns on the 1st and 2nd characteristic individually or in multiple numbers.

  The microreactor according to the first feature includes an automatic control pump for feeding or withdrawing the first fluid, the second fluid, and the amphiphilic molecule mixed solution into the microchannel from each inlet. You may prepare.

  The microreactor according to the second feature may include an automatic control pump for feeding or withdrawing the first fluid and the amphiphilic molecule mixed solution into the microchannel from each inlet.

  In the microreactor according to the first feature, one or more valves may be provided in the microchannel, the first inlet, the second inlet, or the third inlet.

  In the microreactor according to the second feature, one or more valves may be provided in the microchannel, the first inlet, or the third inlet.

  Moreover, you may further provide the branch channel branched from the microchannel of the microreactor which concerns on the 1st and 2nd characteristic, and the inlet which inject | pours a sample.

  Moreover, you may provide an electrode in a part of microchannel of the microreactor which concerns on the 1st and 2nd characteristic.

  Further, a means for controlling the temperature, pressure, pH, current, voltage, magnetic field, ultrasonic wave, or stress in the microchannel of the microreactor according to the first and second features may be further provided.

  In the microreactor according to the first and second features, there is provided means for controlling a temperature change in the microchannel due to light irradiation, a change in adsorption, adhesion, deposition and permeation of a substance to a molecular film, or a photochemical reaction. Further, it may be provided.

  The microreactor according to the first feature may further include one or more salt bridges.

  The third feature of the present invention is that the microchannel, the first inlet for introducing the first fluid into the microchannel, the second inlet for introducing the second fluid into the microchannel, and the amphiphilic property. A first inlet and a third inlet provided between the first inlet and the second inlet for introducing an amphiphilic molecule mixture containing molecules into the microchannel; A microreactor that forms a molecular film at a boundary surface with a fluid, a first fluid, or a second fluid, or a substance contained in the first fluid and the second fluid, and a membrane by interaction with the molecular film A reaction part that causes a change in the surface state of the molecular film or a change in the chemical or physical properties of the molecular film or fluid by the adhesion, adsorption, deposition or permeation of the liquid, and a condition control part that controls the conditions of the reaction part; Detects change signal in reaction area Detection unit, separation / recovery unit that separates and collects the product generated in the reaction unit, circulation unit that circulates fluid and amphiphilic molecules, and production / regeneration unit that creates and regenerates molecular membranes The gist is that the analysis system comprises

  According to the analysis system according to the third feature, automatic, rapid, and continuous molecular membranes such as monolayer, bilayer, reverse bilayer, lipid bilayer, molecular multilayer, reverse micelle, and micelle in the microreactor. A manufacturing method is provided, and various analyzes using this system can be performed.

  The condition control unit of the analysis system according to the third feature may control the temperature, pressure, pH, current, voltage, magnetic field, ultrasonic wave, light, or stress in the microchannel.

  In addition, the detection unit of the analysis system according to the third feature includes detecting a change in temperature, concentration, pH, and electric signal by the interaction between the molecular film and the substance in the microchannel or outside the microchannel. You may observe the surface state change in.

  According to a fourth aspect of the present invention, there is provided a microchannel, a first inlet for introducing a first fluid into the microchannel, a second inlet for introducing a second fluid into the microchannel, and an amphiphilic property. In a microreactor having a first inlet and a third inlet provided between the second inlet and introducing an amphiphilic molecule mixture containing molecules into the microchannel, the first fluid and The step of forming a molecular film at the interface with the second fluid, the interaction between the first fluid, the second fluid, or the substance contained in the first fluid and the second fluid, and the molecular film A step that causes a change in the surface state of a molecular film caused by adhesion, adsorption, deposition, or permeation to the film, or a change in chemical or physical properties of the molecular film or fluid, Action A step for controlling conditions, a step for detecting changes in the surface state of the molecular film, a change in chemical or physical properties of the molecular film or fluid, or a change in the permeation of the substance into the molecular film, and a reaction unit. The gist of the present invention is an analysis method including a step of separating and recovering the produced product, a step of circulating a fluid and an amphiphilic molecule, and a step of producing and regenerating a molecular film.

  According to the analysis method according to the fourth feature, automatic, rapid and continuous molecular membranes such as monolayer, bilayer, reverse bilayer, lipid bilayer, molecular multilayer, reverse micelle and micelle in the microreactor. A manufacturing method is provided, and various analyzes using this system can be performed.

  According to a fifth aspect of the present invention, there is provided a microchannel, a first inlet for introducing a first fluid into the microchannel, a second inlet for introducing a second fluid into the microchannel, and an amphiphilic property. A first inlet and a third inlet provided between the first inlet and the second inlet for introducing an amphiphilic molecule mixture containing molecules into the microchannel; A molecular film is formed at the interface with the fluid, and is contained in the fluid in the microchannel by an enzyme immobilized on the molecular film present in the microchannel or an enzyme immobilized in a certain space of the microchannel. Generated by the enzyme reaction, the reaction part where the enzyme reaction of the substrate is performed, the circulation part that circulates the unreacted substrate and enzyme of the enzyme reaction, the production / regeneration part that produces and regenerates the molecular film on which the enzyme is immobilized Life And separation and recovery unit to separate and recover objects, a gist that the reaction system comprising a detection unit for detecting the result of an enzymatic reaction.

  According to the reaction system according to the fifth feature, automatic, rapid, and continuous molecular membranes such as monolayer, bilayer, reverse bilayer, lipid bilayer, molecular multilayer, reverse micelle, and micelle in the microreactor. In addition to providing a production method, various bioreactors using this system can be performed.

  According to a sixth aspect of the present invention, there is provided a microchannel, a first inlet for introducing a first fluid into the microchannel, a second inlet for introducing a second fluid into the microchannel, and an amphipathic property. In a microreactor having a first inlet and a third inlet provided between the second inlet and introducing an amphiphilic molecule mixture containing molecules into the microchannel, the first fluid and A step of forming a molecular film at the interface with the second fluid, and an enzyme immobilized on the molecular film present in the microchannel or an enzyme immobilized in a constant space of the microchannel. A step of performing an enzyme reaction of a substrate contained in the fluid, a step of circulating unreacted substrate and enzyme of the enzyme reaction, a step of producing and regenerating a molecular film on which the enzyme is immobilized, And separating and recovering the product produced by the enzymatic reaction, is summarized in that a reaction method comprising the step of detecting a result of an enzymatic reaction.

  According to the reaction method according to the sixth feature, automatic, rapid and continuous molecular membranes such as monomolecular membrane, bilayer membrane, reverse bilayer membrane, lipid bilayer membrane, molecular multilayer membrane, reverse micelle and micelle in the microreactor. In addition to providing a production method, various bioreactors using this system can be performed.

  According to a seventh aspect of the present invention, there is provided a microchannel, a first inlet for introducing a first fluid into the microchannel, a second inlet for introducing a second fluid into the microchannel, and an amphiphilic property. A first inlet and a third inlet provided between the first inlet and the second inlet for introducing an amphiphilic molecule mixture containing molecules into the microchannel; A refolding part that forms a molecular film at the interface with the fluid and refolds the structurally abnormal protein and denatured protein contained in the aqueous phase in the microchannel, and separation / recovery that separates and recovers the refolded protein. A recovery unit, a circulation unit for circulating unreacted structurally abnormal proteins and denatured proteins contained in the aqueous phase, a production / regeneration unit for producing / regenerating a molecular film, and refoldin And summarized in that a reaction system comprising a detector for detecting the protein.

  According to the reaction system according to the seventh feature, due to the interaction between the structurally abnormal protein and the denatured protein and the molecular film that is automatically, rapidly and continuously produced in the microreactor, Provide fast and sensitive detection microchips, biosensing systems, or use microreactors equipped with molecular membranes to refold and separate structurally abnormal proteins and denatured proteins in a simple, short time, energy-saving and mild condition ( Extraction), an early diagnosis and treatment system for intractable diseases can be provided.

  According to an eighth aspect of the present invention, there is provided a microchannel, a first inlet for introducing a first fluid into the microchannel, a second inlet for introducing a second fluid into the microchannel, and an amphiphilic property. In a microreactor having a first inlet and a third inlet provided between the second inlet and introducing an amphiphilic molecule mixture containing molecules into the microchannel, the first fluid and A step of forming a molecular film at the interface with the second fluid, a step of refolding the structurally abnormal protein and the denatured protein contained in the aqueous phase in the microchannel, and separating and collecting the refolded protein A step of circulating unreacted structurally abnormal proteins and denatured proteins contained in the aqueous phase, a step of producing and regenerating a molecular film, And summarized in that a reaction method comprising the steps of detecting the Rudingu protein.

  According to the reaction method according to the eighth feature, due to the interaction between the structurally abnormal protein and the denatured protein and the molecular film that is automatically, rapidly and continuously produced in the microreactor, Provide fast and sensitive detection microchips, biosensing systems, or use microreactors equipped with molecular membranes to refold and separate structurally abnormal proteins and denatured proteins in a simple, short time, energy-saving and mild condition ( Extraction), an early diagnosis and treatment system for intractable diseases can be provided.

  A ninth feature of the present invention is that a microchannel in which a pair of electrodes or electrode systems having a constant interval is arranged in the direction of fluid flow, a fluid (61) and an insulating medium (62) into the microchannel. ), A fluid containing a substance (63), an amphiphilic molecule mixed solution containing an amphiphilic molecule (64), a fluid containing a substance (65), an insulating medium (66), and a fluid (67) repeatedly. A microreactor that forms a molecular film on the boundary surface between the fluid (63) and the fluid (65), and adhesion to the film due to the interaction between the substance contained in the fluid (63) and the fluid (65) and the molecular film; Reaction part causing change in surface state of molecular film or chemical or physical property of molecular film or fluid by deposition or permeation, condition control part for controlling reaction part condition, change in reaction part Check the signal A detection portion for, and summarized in that an analytical system comprising a fluid, a prepared and reproducing unit to produce and reproduce circulation unit and molecular membrane circulating amphiphilic molecules and the insulating medium.

  According to the analysis system according to the ninth feature, automatic, rapid, and continuous molecular membranes such as monolayer, bilayer, reverse bilayer, lipid bilayer, molecular multilayer, reverse micelle, and micelle in the microreactor. A manufacturing method is provided, and various analyzes and electrochemical reactions using this system can be performed.

  The analysis system according to the ninth feature is that the fluid in the microchannel, the amphiphilic molecule mixture, and the insulating medium stay in the microchannel in order to maintain the stable state of the formed molecular film. It may flow.

In addition, the insulating medium in the analysis system according to the ninth feature does not transmit ions, has a low dielectric constant, and may clean a fluid, a fluid containing a substance, and electrode contamination caused by a molecular film. .

  In addition, a plurality of pairs of electrodes having a constant interval in the analysis system according to the ninth feature may be arranged in the microchannel.

  The electrode system in the analysis system according to the ninth feature is a two-electrode system, a three-electrode system, or a four-electrode system, and the working electrode and the counter electrode are arranged on the same fluid side, or are separated from each other with a molecular film. May be.

  The fluid (67) in the analysis system according to the ninth feature is a small amount of pure water, organic solvent, sulfuric acid, hydrochloric acid, hydrofluoric acid and nitric acid, or perchloric acid and hydrogen peroxide for cleaning the electrodes. Any of the added acid solution and alkaline solution, and one or more of them may be continuously flowed.

  The electrode pair or electrode system electrode in the analysis system according to the ninth feature may be renewed in the fluid (67) by chemical cleaning or electrochemical cleaning.

  The sample in the analysis system according to the ninth feature is where the sample (63) and the fluid (65) stay, and even if the sample is injected into the fluid (63) or the fluid (65) by the fine channel connected to the microchannel. Good.

  The analysis system according to the ninth feature may further include means for controlling temperature, pressure, pH, current, voltage, magnetic field, ultrasonic wave, light, or stress in the microchannel.

  The microreactor in the analysis system according to the ninth feature may further include a molecular film containing an ion channel or a salt bridge.

The detection unit of the analysis system according to the ninth feature includes the temperature, concentration,
Changes in pH, electrical signals, and changes in the surface state of the molecular film may be detected inside or outside the microchannel.

  A tenth feature of the present invention is a microchannel in which a pair of electrodes or electrode systems having a constant interval is arranged in the direction of fluid flow, a fluid (61) and an insulating medium (62) into the microchannel. ), A fluid containing a substance (63), an amphiphilic molecule mixed solution containing an amphiphilic molecule (64), a fluid containing a substance (65), an insulating medium (66), and a fluid (67) repeatedly. , A step of forming a molecular film on the boundary surface between the fluid (63) and the fluid (65), and adhesion to the film by the interaction between the fluid (63) and the substance contained in the fluid (65) and the molecular film, adsorption, The step of causing the change of the surface state of the molecular film or the chemical or physical property of the molecular film or the fluid by the deposition or permeation, the step of controlling the conditions of the reaction part, and the change signal of the reaction part Detection A step that, fluid, comprising the steps of circulating the amphiphilic molecules and the insulating medium, and summarized in that a method of analysis comprising the steps of preparing and reproducing molecular film.

  According to the analysis method according to the tenth feature, in a microreactor, a molecular film such as a monolayer, a bilayer, a reverse bilayer, a lipid bilayer, a molecular multilayer, a reverse micelle, and a micelle is automatically, rapidly, continuously A manufacturing method is provided, and various analyzes and electrochemical reactions using this system can be performed.

  The eleventh feature of the present invention is a microchannel, a first inlet for introducing a first fluid into the microchannel, a second inlet for introducing a second fluid into the microchannel, and an amphipathic property. A first inlet and a third inlet provided between the first inlet and the second inlet for introducing an amphiphilic molecule mixture containing molecules into the microchannel; A reaction unit that forms a molecular film at the interface with the fluid, interacts with the structurally abnormal protein contained in the aqueous phase in the microchannel or virus and the molecular film, and a detection unit that detects the change signal of the reaction unit And a separation / recovery unit that separates and removes structurally abnormal proteins or viruses by adhesion, adsorption, deposition, or permeation to the molecular film by interaction with the above.

  According to the separation system according to the eleventh feature, due to the interaction between the structurally abnormal protein (amyloid type protein) and the molecular membrane automatically, rapidly and continuously produced in the virus microreactor, Providing a simple, rapid and sensitive detection microchip and biosensing system, or providing an early diagnosis and treatment system for intractable diseases by separating structurally abnormal proteins and viruses using a microreactor equipped with a molecular film it can.

  A twelfth feature of the present invention is a microchannel, a first inlet for introducing a first fluid into the microchannel, a second inlet for introducing a second fluid into the microchannel, and an amphiphilic property. In a microreactor having a first inlet and a third inlet provided between the second inlet and introducing an amphiphilic molecule mixture containing molecules into the microchannel, the first fluid and A step of forming a molecular film at the interface with the second fluid, a step of interacting with a structural abnormal protein or virus in the aqueous phase in the microchannel and the molecular film, and a molecule caused by the interaction Changes in membrane surface conditions, changes in chemical or physical properties of molecular membranes or fluids, or adsorption, attachment, deposition and permeation of structurally abnormal proteins or viruses into molecular membranes A step of detecting a change, attachment to the molecule membrane by interactions, adsorption, by deposition or transmitted, separating the structural abnormalities proteins or viruses, and summarized in that a separation method and removing.

  According to the separation method according to the twelfth feature, the amount of structurally abnormal protein or virus can be traced by the interaction of the structurally abnormal protein (amyloid-type protein) and the molecular membrane that is automatically, rapidly and continuously produced in the virus microreactor. Provide an early diagnosis and treatment system for intractable diseases by providing a simple, rapid and highly sensitive detection microchip and biosensing system, or separating structurally abnormal proteins or viruses using a microreactor equipped with a molecular film Can do.

  The thirteenth feature of the present invention is that the microchannel, the sixth inlet for introducing the reverse micelle solution into the microchannel, the seventh inlet for introducing the raw material aqueous solution containing the substance into the microchannel, and the redox agent A tenth inlet for introducing a reverse micelle solution containing a molecular chaperone, water, and metal ions, and in the microreactor, a substance in the raw water phase is normally extracted into the reverse micelle solution, or a denatured protein accompanying the extraction A normal extraction section where refolding is performed, a raw material recovery section that recovers or recirculates the raw material aqueous solution, a microchannel, an eleventh inlet for introducing the reversely extracted reverse micelle solution into the microchannel, and a recovered aqueous solution A twelfth inlet for introducing the substance into the microchannel, and the substance from the reverse micelle solution extracted in the microreactor in the microreactor, Alternatively, an extraction system comprising a back extraction unit that performs reverse extraction of the refolded protein, a circulation unit that circulates the reverse micelle solution, and a product recovery unit that recovers the back extracted substance or the refolded protein. It is a summary.

  According to the extraction system according to the thirteenth feature, highly efficient and rapid extraction of substances and high activity, high efficiency and rapid extraction of biologically related substances such as proteins with simple, short time, energy saving and mild reaction / extraction conditions. In addition, it is possible to provide a micro-extraction system using reverse micelles for rapid refolding of denatured proteins.

  Further, in the extraction system according to the thirteenth feature, the reverse micelle is a reverse micelle comprising at least one kind of amphiphilic molecule, at least one kind of amphiphilic molecule and at least one kind of co-surfactant, It may be a mixed reverse micelle comprising a ligand, phospholipid, glycolipid, cholesterol, protein, sphingolipid, redox species, cell recognition molecule, and receptor.

  In the extraction system according to the thirteenth feature, the nucleic acid-related substance and / or protein are mixed in the water pool present at the center of the reverse micelle, or a living body related substance is incorporated therein. May be.

  In the extraction system according to the thirteenth feature, the redox agent, molecular chaperone, and metal ion are incorporated into the reverse micelle water pool containing the denatured protein, and the three-dimensional structure and activity of the protein in the reverse micelle water pool. Recovery may be performed.

  In the extraction system according to the thirteenth feature, the back extraction unit includes a branch channel for controlling the pH or salt concentration of the recovered aqueous phase, or a branch channel for adding a hydrophilic organic solvent. You may provide the apparatus which controls temperature conditions.

  The fourteenth feature of the present invention is a microchannel, a sixth inlet for introducing a reverse micelle solution into the microchannel, a seventh inlet for introducing a raw material aqueous solution containing the substance into the microchannel, and a redox agent A tenth inlet for introducing a reverse micelle solution containing a molecular chaperone, water, and metal ions, and in the microreactor, a substance in the raw water phase is normally extracted into the reverse micelle solution, or a denatured protein accompanying the extraction The step of refolding, the step of collecting or recirculating the raw material aqueous solution, the microchannel, the eleventh inlet for introducing the reverse extracted micellar solution into the microchannel, and the recovered aqueous solution into the microchannel. A twelfth inlet for introducing substances from the reverse micelle solution extracted in the microreactor. Or an extraction method comprising a step of back-extracting the refolded protein, a step of circulating a reverse micelle solution, and a step of recovering the back-extracted substance or the refolded protein. To do.

  According to the extraction method according to the fourteenth feature, high-efficiency and rapid extraction of substances and high-activity, high-efficiency and rapid extraction of biological substances such as proteins, with simple, short-time, energy-saving and mild reaction / extraction conditions In addition, it is possible to provide a micro-extraction system using reverse micelles for rapid refolding of denatured proteins.

  A fifteenth feature of the present invention is a cell supply system for supplying cells, a cell-free liquid generation system for generating a cell-free liquid, an amino acid supply system for supplying amino acids, a nucleic acid supply system for supplying nucleic acids, and an energy -Energy supply system that supplies-, cell-free protein synthesis system that synthesizes protein, extraction / refolding system that extracts or refolds the protein synthesized in cell-free protein synthesis system, and cell-free protein synthesis system Or ATP regeneration system that recovers and regenerates ATP from the extraction / refolding system, and a protein recovery system that recovers protein from the cell-free protein synthesis system or from the extraction / refolding system Is the gist.

  According to the protein synthesis system according to the fifteenth feature, a biomimetic tampagne synthesis system can be provided using a microreactor equipped with a molecular film and a microextraction system using reverse micelles.

  The protein synthesis system according to the fifteenth feature is characterized in that the cell-free liquid generation system is a microreactor equipped with a molecular membrane, and electrostatic, hydrophobic, hydrophilic and Intracellular organelles and cytoplasmic substrates may be extracted by ionic interactions, membrane fusion by bioaffinity, or by stimulating molecular membranes.

  Further, in the protein synthesis system according to the fifteenth feature, the cell-free protein synthesis system synthesizes a protein in a microreactor including a molecular film, and the synthesized protein is electrostatically, hydrophobically, and hydrophilically coupled with the molecular film. Furthermore, the protein may be extracted by adhesion, adsorption, deposition, permeation or incorporation into a molecular film by ionic interaction or bioaffinity, and protein denaturation due to protein aggregation may be avoided.

  In the protein synthesis system according to the fifteenth feature, the protein extraction / refolding system may perform protein extraction and refolding in a microreactor having a molecular film or a microreactor extraction system using reverse micelles.

  In the protein synthesis system according to the fifteenth feature, the ATP regeneration system may regenerate ATP by electrochemical reaction, photoelectrochemical reaction, and chemical reaction in a microreactor including a molecular film.

  A sixteenth feature of the present invention is the step of supplying a cell-free solution from a cell supply system to a cell-free solution generation system, extracting and generating the cell-free solution in the cell-free generation system, and cell-free protein synthesis system. A step of synthesizing a protein in a cell-free protein synthesis system by supplying cell-free liquid from the liquid generation system, amino acid from the amino acid supply system, nucleic acid from the nucleic acid supply system, ATP from the ATP generation system, and energy from the energy supply system. Extracting and refolding the protein synthesized by the cell-free protein synthesis system, recovering and regenerating ATP from the extraction and refolding system, and recovering the protein generated by the extraction and refolding system The present invention is summarized as a protein synthesis method including a step.

  According to the protein synthesizing method according to the sixteenth feature, a biomimetic tampagne synthesis system can be provided using a microreactor equipped with a molecular film and a microextraction system using reverse micelles.

  (1) According to the present invention, in the microreactor, monolayer, bilayer, reverse bilayer, lipid bilayer, molecular multilayer, reverse micelle, micelle, vesicle, reverse vesicle, emulsion, LB membrane, etc. Providing automated, rapid, and continuous production methods of molecular membranes, as well as various rapid chemical reactions, bioreactors, structurally and denatured protein refolding, separation (extraction) systems and analysis systems using this system be able to.

  (2) Also, according to the present invention, trace amounts of these substances and viruses can be obtained by interaction with molecular membranes that are automatically, rapidly and continuously produced in structurally abnormal proteins (amyloid-type proteins) or virus microreactors. Provide simple, rapid and sensitive detection microchips, biosensing systems, or perform refolding and separation (extraction) of structurally abnormal proteins using a microreactor equipped with molecular membranes, enabling early diagnosis and treatment systems for intractable diseases Can be provided.

  (3) In addition, according to the present invention, high-efficiency and rapid extraction of substances, high-activity, high-efficiency and rapid extraction of biological substances such as proteins, under simple, short-time, energy-saving and mild reaction / extraction conditions, A micro-extraction system using reverse micelles for rapid refolding of denatured proteins can be provided.

  (4) Moreover, according to the present invention, a biomimetic tampane synthesis system can be provided by using a microreactor equipped with a molecular film and a microextraction system using reverse micelles.

  Next, embodiments of the present invention will be described with reference to the drawings. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, it should be noted that the drawings are schematic and ratios of dimensions and the like are different from actual ones. Accordingly, specific dimensions and the like should be determined in consideration of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.

(First embodiment)
In the first embodiment, monolayer, bilayer, lipid bilayer, reverse bilayer, molecular multilayer, lipid multilayer, micelle, reverse micelle, vesicle, reverse vesicle, emulsion, LB membrane A microreactor capable of automatically, rapidly and continuously producing a molecular film having a structure in which any one or a mixture of these will be described. Various rapid chemical reactions using this microreactor, bioreactors, refolding of structurally abnormal proteins and denatured proteins, separation (extraction) systems, and biosensing systems (microanalysis systems) will be described.

(Characteristics of the microreactor according to the first embodiment)
In the first embodiment, a stable contact interface between a liquid laminar flow / liquid laminar flow, a gas phase / liquid laminar flow, and a liquid laminar flow / solid phase formed in a microchannel is used. Along with the laminar flow direction between the interfaces, the amphiphilic molecule mixture for producing the molecular film is continuously or intermittently discharged, thereby having a large area and a different shape, which have been conventionally difficult, It is characterized by producing a stable molecular film that can be easily updated.

  Further, the present invention is characterized by realizing a microreactor based on the permeation of various substances, chemical reactions, and the interaction between films and substances by utilizing the characteristics of the molecular film existing in the microspace.

  In particular, the present inventors have found that a lipid bilayer membrane formed in a microchannel can be used as a biological membrane model, and completed the present invention.

  That is, in the first embodiment, the microreactor is provided with a molecular film, and the microreactor has a third inlet for introducing an amphiphilic molecule mixed solution, a first phase for introducing an aqueous phase, an oil phase, or a gas phase. An introduction port or a second introduction port is provided, and these introduction ports have a ring shape, an elliptical shape, a semicircular shape, a quadrangular shape, and an unequal side polygonal shape.

  The microreactor according to the first embodiment includes at least one kind of protein, nucleic acid, glycolipid, cholesterol, fluorescent dye, ligand, photosensitive molecule, ion channel, electron conjugated substance, co-surfactant, crown Ether, fullerene, carbon nanotubes, porphyrins, cyclodextrins, molecular tongs, redox agents, steroids, polypeptides or peptides, including at least one amphiphilic molecule, lipid, sphingolipid, boundary lipid And a third inlet 21 for introducing an amphiphilic molecule mixed solution 25 composed of a lipid modified with a fluorescent molecule and an organic solvent, and a first fluid 26 and a second fluid containing biological substances and various substances. 27 is provided with a first inlet 22 and a second inlet 23, and one of these inlets is the above-mentioned both An infusion channel of the amphiphilic molecule mixture 25, the first fluid 26 and the second fluid 27 and a pump having an automatic control function are connected, and the other is connected to a microchannel having various three-dimensional shapes. Between the laminar flow of the first fluid 26 and the second fluid 27 or between the first fluid 26 and the second fluid 27 and the wall surface of the microchannel, two-dimensionally penetrating the microchannel made of an amphiphilic molecule mixture. (Planar) or a three-dimensional (cylindrical, elliptical cylindrical, etc.) structure and a molecular film containing molecules and molecules are formed automatically, rapidly and continuously. In the case of a bilayer membrane, it functions as an artificial biological membrane model, providing a field for material permeation and chemical reaction or a boundary between both fluids in contact with both sides. It has the function to control the experimental conditions of temperature, pressure, pH, current, voltage, magnetic field, ultrasonic wave, or stress in the channel, or the incident of light into the channel, the substance to the molecular film accompanying the light irradiation It has the function of controlling adsorption, deposition, deposition and permeation and chemical reaction, and further comprises control and detection means for controlling and detecting various chemical reactions occurring in the microchannel, or the first fluid 26 And adhesion of one or more substances contained in the second fluid 27 to the film by the interaction with the molecular film body, adsorption and deposition, and permeation of these substances into the film, or the first fluid 26 and the second fluid And a device capable of detecting the release behavior of the substance contained in the endoplasmic reticulum by fusing with the molecular membrane of the endoplasmic reticulum present in the fluid 27.

  The molecular film in the first embodiment is a biological material, a monomolecular film comprising a molecule, a bimolecular film, a lipid bimolecular film, an inverted bimolecular film, a molecular multilayer film, a lipid multilayer film, a micelle, Any one of reverse micelles, vesicles, reverse vesicles, emulsions, and LB films, or a structure in which these are mixed are characterized in that they exist.

  In addition, the molecular film in the first embodiment has at least one of a two-dimensional planar shape, a three-dimensional columnar shape, a conical shape, a truncated conical shape, a cylindrical shape, an elliptical cylindrical shape, and a polygonal shape. It is characterized by.

  Furthermore, the molecular film in the first embodiment is a lipid, boundary lipid, lipid modified with a fluorescent molecule, protein (enzyme, molecular chaperone, antigen, antibody), nucleic acid, glycolipid, cholesterol, fluorescent dye, ligand, sphingolipid. , Photosensitive molecules, ion channels, electron conjugated substances, co-surfactants, crown ethers, fullerenes, carbon nanotubes, porphyrins, cyclodextrins, molecular tongs, polypeptides or peptides. It is a feature.

  The first introduction port 22, the second introduction port 23, and the third introduction port 21 in the first embodiment are linked, elliptical, angular, hexagonal, unequal side polygonal, and the like. It is a feature.

  In addition, the 3rd inlet 21, the 1st inlet 22, and the 2nd inlet 23 do not need to be designed on the same plane.

  The temperature of the microreactor in the first embodiment includes a system capable of controlling the temperature of the entire system by light irradiation into the microchannel, a hot plate, a thermostat, or a micro temperature control chip integrated with the macro reactor. It is characterized by doing.

  As detection means for detecting various chemical reactions occurring in the microchannel and material permeation into the molecular film in the first embodiment, the molecular film formed in the microchannel, the aqueous phase, the oil phase, and the gas phase Measurement of electrical signal change caused by interaction with substance components contained in the first fluid 26 and second fluid 27, measurement of pH, viscosity, density, refractive index, absorption spectrum change and fluorescence spectrum change , Detection of fluorescent substances, ions, redox species and molecules that have passed through the molecular membrane, observation of surface state changes in the molecular membrane, or in the aqueous and oil phases (first fluid 26 and second fluid 27) It is characterized by detecting various change signals by measuring temperature changes.

  The water phase, oil phase, or gas phase (first fluid 26 and second fluid 27) in the first embodiment is an aqueous medium, an organic medium or gas, a supercritical fluid, and is a protein, nucleic acid, or nucleic acid-related substance. Biomaterials such as amino acids, ligands, polypeptides, peptides and sugars, or non-biological materials such as substrates, inorganic substances, fluorescent substances, organic substances, and at least one of these bio-related substances or non-biological substances It contains at least one or more vesicles (depending on the characteristics of the water phase, oil phase, or gas phase).

  The first fluid 26 and the second fluid 27 in the first embodiment are water and water, oil and oil, gas and gas, gas and water, water and supercritical fluid, gas and oil, water and oil, etc. The first fluid 26 and the second fluid 27 are combined in the above.

  When a valve is installed at the inlet and outlet of the microchannel of the first fluid 26, the second fluid 27, and the amphiphilic molecule mixture 25 in the first embodiment, and a stable molecular film is formed, Providing a static solution pool by closing valves at both ends simultaneously or by controlling the inflow pressure of the first fluid 26, the second fluid 27, and the amphiphilic molecule mixture flowing from the inlet. It is characterized by being able to.

  As one of the means for detecting permeation and chemical reaction in the first embodiment, an electrical signal can be detected inside the microchannels on both sides with the molecular film as a boundary, or in the first fluid 26 and the second fluid 27. A microelectrode or a mirror surface capable of reflecting incident light is provided.

  The width, length and shape of the single microchannel constituting the microreactor in the first embodiment are variable, and the single microchannel is composed of one or more types of three-dimensional structures.

  The microreactor including the molecular film according to the first embodiment is configured and integrated from a single microchannel or a multimicrochannel.

  The chemical field in the first embodiment is a fluid on the opposite side of a product of a chemical reaction such as an enzymatic reaction or protein synthesis performed in the first fluid 26 and the second fluid 27 with a molecular film as a boundary. It is characterized by being extracted.

  In the first embodiment, the molecular membrane, in particular, the lipid bilayer membrane that is a biological membrane is accompanied by fluctuations in the membrane when performing membrane fusion with a vesicle or a biological substance having a biological membrane in cell fusion. In particular, the fluctuation of the film is reproduced in the microreactor.

  The driving force that diffuses and permeates the substance into the molecular film 5 has, for example, a concentration gradient or a potential gradient. That is, the substance diffuses and moves by utilizing the natural diffusion due to the concentration gradient of the substance and the promotion of the mass transfer due to the potential gradient caused by the voltage applied to both sides of the membrane.

  Moreover, the substance can permeate the membrane by the following method.

  1) Direct permeation: Directly permeates the molecular membrane (lipid bilayer membrane) (hydrophobic substance such as organic matter).

  2) Membrane permeation through ion channels (various ions).

  3) Membrane permeation (taste substance) by membrane receptors.

  3) Using a technique of electroporation, a small hole is made in the lipid bilayer membrane by electrical stimulation, and a substance (a nucleic acid-related substance such as DNA or RAN, protein, etc.) is sent to the opposite side of the membrane.

  In the first embodiment, the bimolecular film formed in the microchannel can be polymerized.

  In addition, a bimolecular film in which microdomains are partially formed in the microchannel can be formed. Alternatively, a bilayer film having a microdomain-like function can be formed between laminar flows. In particular, in the latter case, since the entire membrane has microdomain characteristics, it can act with sensitivity to substances such as amyloid-type proteins and viruses far more than biological membranes. Therefore, when the microsensor having the latter microdomain-like film is used, the sample can be detected with higher sensitivity and speed.

  The above microdomain-like film is not limited to the microchannel, and even when it is made on a substrate having micropores in the solution, it has the property of interacting with the sample in the same way. When a membrane is constructed, it can be similarly applied as a sensor for amyloid protein or virus.

(Microreactor material, fabrication method and size)
The material constituting the microreactor 1 and the microchannel 3 according to the first embodiment is not particularly limited as long as at least one material having insulating properties is used.

  For example, Pyrex (registered trademark) glass, quartz glass, silica, polymer, ceramics, SiC ceramics, gel, photoresist, plastic, and silicon resin can be used. Moreover, these materials can be joined by a method such as melting and anodic joining, and an insulating material made of a multilayer material can also be used.

  The microchannel can be produced by a template method or the like. Further, it can be produced by a chemical or electrochemical method. In addition, microchannels made of different materials can be manufactured by welding, anodic bonding, or bonding using the above insulating materials. Furthermore, by using a photoreactive photoresist or a thermoplastic plastic, a microchannel can be produced by a method such as light irradiation or mold heating.

  A microreactor can also be configured by bonding a microchannel manufactured by a mold method or the like to a substrate made of a different material.

  As described above, there are various materials and methods for fabricating the microreactor, but the present invention is not particularly limited to these materials and fabrication methods.

  The microchannel 3 is a groove of several tens of μm to several thousand μm, and the flow channel substrate on which the microchannel 3 is formed can use a microchip of several centimeters to several tens of centimeters. In particular, the size is not limited to the above.

(Molecular film type)
The molecular film 5 according to the first embodiment flows two-dimensionally or three-dimensionally between the liquid / liquid laminar flow, the gas / liquid laminar flow, or the liquid laminar flow / wall surface in the microchannel. Static and molecular membrane is any of monolayer, bilayer, lipid bilayer, reverse bilayer, molecular multilayer, lipid multilayer, micelle, reverse micelle, vesicle, reverse vesicle, emulsion, LB membrane Or a structure in which these are mixed.

  The molecular film 5 is composed of at least one kind of amphipathic molecule, lipid, boundary lipid, sphingolipid and lipid modified with a fluorescent molecule, or protein (enzyme, molecular chaperone, antigen, antibody, hormone), nucleic acid, molecule , Glycolipids, cholesterol, fluorescent dyes, ligands, photosensitive molecules, ion channels, electron conjugated substances, co-surfactants, crown ethers, fullerenes, carbon nanotubes, porphyrins, cyclodextrins, molecular tongs, redox agents A membrane containing at least one substance such as a substance such as steroid, polypeptide or peptide.

  For example, lipids can be used as the amphiphilic molecules constituting the molecular film. Examples of lipids that form lipid bilayers include triolein, monoolein, egg yolk lecithin, phospholipids, synthetic lipids, lysophospholipids, glycosyl diacylglycerols, plasmalogens, sphingomyelins, gangliosides , Fluorescent lipids, sphingolipids, glycosphingolipids, lecithins, sterolites, sterols, cholesterol, oxidized cholesterol, sihydrocholesterol, glyceryl distearate, glyceryl monooleate, glyceryl dioleate, isosorbate monobradede, sorbitan Tristearate, sorbitan monooleate, sorbitan monopalmitreato, sorbitan monolaurate, sorbitan monobrushdate, dodecylic acid / phosphate, dioctade Phosphate, tocophenol, chlorophyll, xanthophylls, phosphatidylethanolamine, phosphatidylserine, inositol, hexadecyltrimethylammonium bromide, diglucosyl diglyceride, phosphatidylcholine, retinal / cholesterol oxide / lectin / rhodopsin, brain Total lipids, human erythrocyte total lipids, and the like can be used, but other lipids and synthetic lipids that can form a molecular film are not particularly limited.

  In addition, as a solvent for dissolving amphiphilic molecules (lipids) in order to produce a molecular film, for example, C4 to C6 hydrocarbons, C6 to C12 hydrocarbons, decane, octane, dodecane, hexane, butanol, hexanedecane Hydrophobic solvents such as chloroform and hydrophilic solvents such as methanol and acetonitrile can be used. Usually, it is desirable to use a nonpolar solvent such as decane rather than a polar solvent such as chloroform. The above-mentioned solvents can be used alone or in combination, and a small amount of water may be added to the solvent in order to dissolve the water-soluble biological substance. As long as the solvent and the mixed solvent can dissolve and disperse the amphiphilic molecule (lipid), the type of the solvent, the mixing ratio, and the mixed state are not particularly limited.

  In addition, there are cationic surfactants, anionic surfactants, amphoteric surfactants, and nonionic surfactants as surfactants for forming reverse micelle type molecular membranes that are a kind of molecular membranes. Specific examples include the following.

  For example, alkyl quaternary ammonium salts (CTAB, TOMAC, etc.), alkyl pyridinium salts (CPC, etc.), dialkyl sulfosuccinates (AOT, etc.), dialkyl phosphates, alkyl sulfates (SDS, etc.), alkyl sulfonates, polysulfonates, Examples include, but are not limited to, oxyethylene surfactants (Tween, Brij, Triton, etc.), alkyl sorbitans (Span, etc.), lecithin surfactants, betaine surfactants, etc. Absent.

  Further, the solvent constituting the reverse micelle solution is not particularly limited. However, when an organic solvent is used, for example, aliphatic hydrocarbons such as hexane, heptane, octane, isooctane and decane, and aromatic hydrocarbons such as benzene and toluene. , Cycloaliphatic hydrocarbons such as cyclohexane, halogenated aliphatic hydrocarbons such as chloroform and dichloroethane, and the like can be used.

  Moreover, a reverse micelle solution can be prepared using, for example, a supercritical fluid or a high-pressure fluid instead of an organic solvent. For example, a supercritical fluid such as ethane, propane, carbon dioxide, or a high-pressure fluid can be used, but it is not particularly limited thereto.

  In addition, an auxiliary surfactant may be added to stabilize the reverse micelle. Examples include higher alcohols such as octanol, but are not limited thereto.

  In addition, examples of biological substances contained in the molecular membrane include glycolipids, proteins, polypeptides, peptides, nucleic acids, nucleic acid-related substances, and amino acids.

  Proteins therein can be further classified into enzymes, glycoproteins, molecular chaperones, myoglobin, hemoglobin, ATPase, antigens and antibodies. Hereinafter, proteins such as enzymes, antigens, and antibodies used in bioreactors and biosensors according to the present invention will be described in particular, but the present invention is not particularly limited thereto.

  Enzymes incorporated between lipid molecules in molecular membranes, electrostatic, hydrophilic, hydrophobic and ionic interactions on the surface of molecular membranes, enzymes immobilized on bioaffinity by tees, microchannels As an enzyme existing in a certain space, an oxidoreductase, a transferase, a hydrolase, a desorption enzyme, an isomerase, and a synthase can be used. Although these types are shown below, the enzyme used for this invention is not limited to these.

  For example, acyl-CoA oxidase, acyl-CoA synthetase, ascorbate oxidase, aspartate aminotransferase, aspartate β-decarboxylase, aspartase, acetate kinase, aminoacylase, amino acid oxidase, aminopeptidase, amylase, alanine dehydrogenase Arabanase, arabinosidase, RNA polymerase, alkaline xylanase, alkaline cellulase, alkaline protease, alkaline lipase, alcohol dehydrogenase, aldehyde dehydrogenase, aldolase, α-acetolactic decarboxylase, α-chymotrypsin, isoamylase, isocitrate dehydrogenase, invertase , Uricase, urease, urokinase, esterase, N-acetylneuramin Lyase, endo-β-glucanase, ω-hydroxylase, catalase, carboxylesterase, carboxypeptidase, carbonic anhydrase, γ-glutamine transpeptidase, xanthine oxidase, formate dehydrogenase, xylanase, xylan acetylesterase, xylose isomerase, chymosin, guanosine 5'-phosphate synthetase, citrate synthetase, glycerol oxidase, glycerol kinase, glycerol 3-phosphate oxidase, glucoamylase, glucosidase, glucosyltransferase, glucose isomerase, glucose-1-oxidase, glucose oxidase, glucose dehydrogenase, glucose Dehydrogenase, glucose 6-phosphate dehydrogenase , Glutamate decarboxylase, glutamate dehydrogenase, creatininase, creatinine deiminase, creatinase, chloroperoxidase, 5'-adenylate deaminase, colipase, cholinesterase, choline oxidase, cholesterol oxidase, thermolysin, sarcosine oxidase, sarcosine dehydrogenase , 3α-hydroxysteroid dehydrogenase, 3-chloro-D-alanine chloride, diaphorase, cyanate aldolase, cyclodextrin glucosyltransferase, dihydropyrimidinase, streptokinase, superoxide dismutase, subtilisin, cephalosporin acylase, cephalosporin Amidase, cellulase, cellobiohydrolar , Cytochrome C, thymidylate synthase, DNA polymerase, deoxyribose-5-phosphate aldolase, dextranase, dopa decarboxylase, transglutaminase, triose phosphate isomerase, trypsin, tryptophanase, tryptophan synthetase, naringinase, nitrile hydra Tase, lactate dehydrogenase, neuraminidase, halohydrin epoxidase, halohydrin halogen halide lyase, haloperoxidase, histidine ammonia lyase, hydantoinase, pyranose-2-oxidase, pyruvate oxidase, phenylalanine ammonia linase, phenol oxidase, putre Synoxidase, flavin enzyme, purine nucleoside phosphorylase, pullulanase, Rotase, prourokinase, proteinase, proline iminopeptidase, bromoperoxidase, hexokinase, pectinase, pectinesterase, pectin transeliminase, β-etherase, β-galactosidase, β-glucanase, β-glucoamylase, β-fructofuranosidase, β-fructofuracinase, peptidase, hemicellulase, penicillin amylase, penicillin amidase, peroxidase, pentosanase, phosphodiesterase, phospholipase, phosphorylase, polygalacturonase, mannanase, mutanase, mutarotase, lactase, lactonohydrolase, lactoperoxidase, lacto , Racemase, laccase, lyase, ligase, lignin peroxy Enzymes such as oxidase, lysyl endopeptidase, lysine oxidase, lysine decarboxylase, lysozyme, lipase, ribulose 1, 5-bisphosphate carboxylase, lipoprotein lipase, ribonuclease A, malate dehydrokinase, luciferase, leucine aminopeptidase, rhodanase However, it is possible to use artificial enzymes newly created by gene recombination, but are not limited thereto.

  As amino acids, glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, glutamic acid, etc. are used. However, the present invention is not limited to this.

  Examples of peptides include lysyl lysyl glycyl glutamic acid, cysteyl seryl asparaginyl osyl seryl tyrosyl seryl valyl leucyl glycyl lysine, tryptophanyl glycine, tryptophanyl glycyl glycine, tryptophanyl phenylalanine, trypto Fanyltryptophan, phenylalanylglycine, phenylalanylglycylglycine, glycyltyrosine, tyrosylglycylglycine, glycylglycine, glycylglycylglycine, tryptophanylglycine, phenylalanylglycine, tyrosylglycine, phenylalanylvaline , Tyrosylvaline, leucylglycylglycine, glycylserylphenylalanine, glycylphenylalanylserine, tyrosylglycylglycine, glycylphenylalani Alanine, glycylalanyl tyrosine, glycylphenylalanylphenylalanine, glycyllysylphenylalanine, lysylserylphenylalanine, phenylalanylphenylalanine, phenylalanylphenylalanylphenylalanine, lysyllysyltyrosine, tyrosyltyrosylphenylalanine, tyrosyltylo Siltyrosine, alanyltryptophan, glycylphenylalanylphenylalanine and the like are used, but are not particularly limited thereto.

  In addition, as a protein, it begins with rhodopsin that receives light information from the eye, and it detects, transmits, processes and stores information such as immunoglobulins, antibodies and other proteins that protect the body from diseases, hormones, various tastes and smells in the outside world, etc. Proteins, digested proteins, transport proteins, receptor proteins, etc., and humans that exist in nature, regardless of whether they are derived from living organisms, etc. It can also be used.

  In addition, as molecules sensitive to light that can be introduced into the molecular film, for example, those having various π-electron chromophores such as azobenzene, stilbene, anthracene, naphthalene, pyrene, and carbazole can be used. In addition, as a molecule capable of photoinduced electron transfer reaction, for example, a molecule such as tris (2,2′bipyridyl) ruthenium dichloride can be used. Moreover, as a derivative that performs photoisomerization, for example, optical spiropyran can be used. In addition, photosensitive molecules such as rhodopsin, bacteriorhodopsin, chlorophyll, and chlorophyll can be used, but are not particularly limited thereto.

  Examples of nucleic acid-related substances include adenine, guanine, cytosine, thymine, uracil, adenosine triphosphate, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA), but are not particularly limited thereto.

  Examples of organic substances that permeate the lipid bilayer include, for example, indole-3-ethanol, indole, 5-hydroxyindole, serotonin, tryptophan, procaine, p-aminobenzoic acid, salicylic acid, sulfanilic acid, sulfanilamide, 3, 4-dihydroxyphenylalanine, pyridoxamine, pyridoxine, quinidine, quine, acetylcholine, salicylamide, erythritol, mannitol, sorbitol, glucose, D-mannose, D-ribose, D-fructose, 2-deoxy-D-glucose, 3-O-methyl Although -D-glucose and the like are used, it is not particularly limited to these.

(Micro analysis system)
Next, lipids modified with proteins (enzymes, antibodies), nucleic acids, sphingolipids and fluorescent molecules on monolayers, bilayers, lipid bilayers, reverse bilayers and molecular membranes formed in the microphone channel, Incorporates glycolipids, cholesterol, fluorescent dyes, ligands, photosensitive molecules, co-surfactants, crown ethers, fullerenes, carbon nanotubes, porphyrins, cyclodextrins, molecular tongs, redox agents, steroids, polypeptides and peptides The biosensor chip and microreactor analysis system (biosensing system) will be described with reference to FIGS. 1 and 6 to 8.

(Structure of micro analysis system)
As shown in FIG. 1, the micro-analysis system according to the first embodiment of the present invention has a condition control for controlling reaction conditions of various reactions occurring in the production / regeneration unit 11 for producing a molecular film and the microreactor 1. Unit 12, detection unit 13 for detecting and observing the results of these reactions and changes, reaction unit 14 for causing various chemical reactions, physical and chemical phenomena, circulation unit 15 for increasing the efficiency of the reaction, And a separation / recovery unit 16 for recovering the product. These components can be freely combined according to the types of bioreactors and biosensors.

  As shown in FIG. 2, the preparation / regeneration unit 11 includes a third inlet 21 for introducing the amphiphilic molecule mixed solution 25 into the microchannel 3 and the first fluid 26 into the microchannel 3. A first introduction port 22 to be introduced and a second introduction port 23 to introduce the second fluid 27 into the microchannel 3 are provided. In the microchannel 3, when the first fluid 26 and the second fluid 27 are diffused with the amphiphilic molecule mixed solution 25 as a boundary wall, only the molecular film remains at the interface between the two liquids. As shown in FIG. 2A, different molecular films are formed depending on the types of the amphiphilic molecule mixed solution 25, the first fluid 26, and the second fluid 27. For example, a monomolecular film is formed at the water phase (W) / oil phase (O) and gas phase (V) / water phase (W) interfaces, and at the oil phase (O) / oil phase (O) interfaces, A molecular film is formed, and a bimolecular film is formed at the water phase (W) / water phase (W) interface. In addition, molecular film structures such as micelles, reverse micelles, vesicles, reverse vesicles, and emulsions are formed.

  Further, the microchannel 3 may be any one of a straight pipe, an elbow, a bend, or a structure in which these are mixed.

  Further, the wall surface of the microchannel 3 may be any one of a smooth shape, an uneven shape, a surface modification, or a structure in which these are mixed.

  The microchannel 3 may have a groove shape formed in the microreactor 1 as shown in FIG. 2, or may have a ring shape as shown in FIG. In addition, it may have a polygonal shape such as an elliptical shape, a rectangular shape, or a hexagonal shape, depending on conditions such as the use, the type of lipid, and the constituent components of the molecular membrane. Further, depending on the shape of the microchannel 3, the molecular film to be produced has a planar shape or a cylindrical shape. FIG. 2B is a schematic diagram of a planar bimolecular film, and FIG. 4B is a schematic diagram of a cylindrical bimolecular film 35.

  Further, the microreactor 1 includes an automatic control pump for feeding or withdrawing the first fluid 26, the second fluid 27, and the amphiphilic molecule mixed solution 25 into the microchannel 3 from the respective inlets. Further, the microreactor 1 includes one or more valves in the microchannel 3 or the first inlet 22, the second inlet 23, or the third inlet 21. The microreactor includes a branch channel that branches from the microchannel 3 and an inlet for injecting a sample.

  The microchannel 3 of the microreactor 1 is provided with a salt bridge.

  Further, as shown in FIG. 3, the production / regeneration unit 11 includes the microchannel 3, the first introduction port 22 for introducing the first fluid 26 into the microchannel 3, and the amphipathic substance including amphiphilic molecules. A third inlet 21 for introducing the molecular mixed liquid 25 into the microchannel 3 may be provided, and a molecular film may be formed on the boundary surface between the first fluid 26 and the wall surface of the microchannel.

  Further, by integrating the single-tube microchannels shown in FIG. 5A, the multi-tube microchannel shown in FIG. 5B can be provided. In the first embodiment, the system scale-up method is not particularly limited as long as it has a microreactor function having a similar molecular film.

  Note that the shape of the third inlet 21 and the structure of the injection channel connected to the third inlet 21 are not particularly limited as long as a stable, stationary or fluid molecular film can be produced. As shown in FIGS. 2 and 3, the first inlet 22 and the second inlet 23 are connected to independent injection channels, respectively, and the first fluid 26 made of fluids of different compositions and types, The second fluid 27 can flow out.

  The first introduction port 22, the second introduction port 23, and the third introduction port 21 have a semicircular shape (FIG. 2) and a ring shape and a cylindrical shape (FIG. 4), and the first introduction port. 22. Connect to the same microchannel structure (FIG. 2) corresponding to the shape of the second introduction port 23 and the third introduction port 21, respectively, or to the microchannel that gradually changes the microchannel structure. Can do. In addition, the first inlet 22 having a polygonal shape such as an elliptical shape, a rectangular shape, a hexagonal shape, and the like, depending on the use, the type of amphiphilic molecule (lipid) and the constituent components of the molecular film. Two inlets 23 and a third inlet 21 or microchannels of various shapes can be used. In the present invention, the shape of the first inlet 22, the second inlet 23, and the third inlet 21 is not particularly limited as long as a stable and stationary or fluid molecular film can be produced. . Further, the structure of the microchannel connected to the first introduction port 22, the second introduction port 23, and the third introduction port 21 is not particularly limited.

  The condition control unit 12 refers to a part such as an electrode and a device that can be appropriately controlled so that various chemical reactions, physical phenomena, or chemical phenomena occurring in the microreactor 1 can be performed under optimum conditions. These devices and electrodes are provided inside the microreactor 1 or outside the microreactor 1 in order to appropriately control conditions such as temperature, pressure, pH, current, voltage, magnetic field, ultrasonic wave, light, or stress inside the microreactor 1. Can be installed or used in combination.

  For example, devices such as a function generator, an oscilloscope, a potentiostat, and electroporation can be used as a device for controlling the voltage, which is one of the condition control units. However, the molecular film has different strengths and patterns (for example, continuous There is no particular limitation as long as a voltage of a triangular wave, a step wave, a single rectangular wave, a continuous rectangular wave, a ramp wave, a sine wave, etc.) and a frequency (frequency) can be applied.

  For example, when the microreactor 1 has a double cylindrical structure as shown in FIG. 4A, an electrode is provided in the cylindrical portion inside the microreactor 1, and the condition control unit 12 can control this electrode.

  The detection unit 13 observes the molecular film using the measurement device and electrodes listed below, but is not particularly limited thereto.

  As an observation device of the film surface state, an optical microscope, an electron microscope, a laser electron microscope, a differential interference phase contrast microscope, a scanning electron microscope (SEM), a Fourier transform infrared analyzer, a laser Raman spectrometer, a fluorescence microscope, a polarization microscope An analyzer such as a microscope, an X-ray analyzer and an atomic force microscope (AFM) can be used, but is not particularly limited to these measuring devices.

  Also, as the characteristics of the molecular film, for example, physical properties such as film fluidity, packing state, charge density on the film surface, hydrophobic and hydrophilic characteristics, film potential, film impedance, film or solution dielectric constant measurement Can be examined. Devices that measure electrical signals such as membrane potential, voltage, and current include electrometers, digital multimeters, potentiostats, function generators, oscilloscopes, etc., but high resistance, low-pass filters, amplifiers that can amplify signal electrical signals, etc. It is desirable to use a high sensitivity electrometer with As an apparatus for measuring the impedance and capacitance of the membrane, an impedance analyzer, an LCR meter, a vector impedance meter, and the like can be used, but a potentiostat, in particular, a signal electrical signal smaller than a picoampere can be measured and a high-speed sweep can be performed. It is desirable to use in combination with a device such as an ultra-sensitive potentiostat equipped with a low-pass filter. As another electrochemical measuring apparatus, a bipotentiostat (dual potentiostat) can be used. In order to prevent electrical noise, it is desirable to perform the above measurement in a shield box.

  On the other hand, biologically related substances such as polypeptides, saccharides, proteins, and viruses are attached to the membrane due to the interaction with the membrane. An observation device can be used.

  Furthermore, substances such as organic substances, saccharides, amino acids and ions permeate through membranes, or substances such as polypeptides, saccharides, bitter substances, sweet substances, umami substances, odor substances, volatile substances and proteins and viruses interact with the membrane. It is possible to detect and observe indirectly or directly the change in the permeation of substances such as ions and molecules in the solution phase due to the change in the film, the change in the electrical signal, or the change in the characteristics of the film.

  In addition, various changes in signals caused by the interaction between a fluid or a substance contained in the fluid and the molecular film, or a method for directly and indirectly detecting ions, molecules and substances that have passed through the molecular film. This will be described below.

  The direct detection method is a method for directly observing and detecting various reactions and signal changes caused by the molecular film on the spot in the microchannel.

  Signal changes include, for example, electrical signals of molecular films and fluids, changes in signals such as pH, viscosity, density, refractive index, absorption spectrum, fluorescence spectrum and heat, or changes in signals related to the surface state of molecular films, etc. Is raised.

  For example, an electrode such as an ion sensor, a microelectrode, or a sensor electrode that is a sensor terminal of an electrometer, a potentiostat, a function generator, an oscilloscope, and a galvanostat device is installed in the microchannel. These electrodes can be detected locally and in-situ within the microchannel.

  Changes in the pH, viscosity, density and refractive index can be measured with a pH meter, a viscometer, a density meter, and a refractometer.

  Changes in the absorption spectrum and fluorescence spectrum signals can be detected by, for example, introducing and transmitting the corresponding light source directly into the microchannel and measuring the transmitted light and fluorescence changes.

  Indirect detection methods include, for example, collecting the relevant fluid segments at the outlet of the microchannel and changing the signals such as electrical signal, pH, viscosity, density, refractive index, absorption spectrum, fluorescence spectrum and heat of each segment. Is a method of detecting

  In an indirect detection method, depending on the characteristics of the substance to be detected contained in each section flowing out from the microchannel and the purpose of detection, etc., potentiostat, pH meter, viscometer, density meter, refractometer, absorbance Photometer, Fluorescence Spectrometer, High Performance Liquid Chromatography (HPLC), Liquid Chromatography (LC), Gas Chromatography (GC), Gas Chromatography Mass Spectrometer (GC / MS), Thin Layer Chromatography Analyzer (TLC) A chemical analyzer such as a Fourier transform infrared spectrometer (FTIR) can be used as appropriate.

  In summary, if the substance to be detected is not a fluorescent substance, it can be qualitatively and quantified by measuring the electric signal, pH, viscosity, density, refractive index, absorption spectrum and thermal signal of the substance. When the substance to be detected is a fluorescent substance, it can be qualitatively and quantitatively detected by detecting a change in the fluorescence spectrum.

  For analysis of peptides, polypeptides and proteins, high performance liquid chromatography (HPLC), spectrophotometry and circular dichroism spectroscopy can be used. The secondary structure of a protein can be analyzed using Fourier transform infrared spectroscopy.

  In the first embodiment, as a detection of a substance that has passed through a molecular film in a microreactor or a substance newly generated by a chemical reaction, detection is performed using a sensor electrode or a microelectrode appropriately disposed in the microchannel. Can do. These electrodes are not particularly limited as long as those substances can be detected. However, in order to detect only the target substance with high sensitivity, an electrode capable of selective detection is preferable. For example, one method is a method using an ion selective electrode. This detects membrane potential fluctuations due to the difference in concentration of ions to be measured in an ion selective membrane that responds to specific ions. Depending on the type of membrane used, halogen ions such as hydrogen ions, iodine ions, and bromide ions are used. Various ions can be detected. These electrodes are usually prepared by immobilizing an ion-selective membrane directly or via an internal standard solution on the surface of a reference electrode such as a refined saturated calomel electrode or a silver / silver chloride electrode, or A minute ion selective FET electrode in which an ion selective film is disposed on a gate insulating film of a microfabricated field effect transistor (FET) can be used.

  In addition to the potential measurement using the ion-selective membrane described above, a method for detecting a current associated with redox of a target atom, molecule, or redox species such as those ions can also be used. In this case, platinum, gold, carbon, or the like is usually used as the substance constituting the electrode from the viewpoint of chemical stability and reaction activity on the surface, but depending on the detection substance in the medium used, platinum- Carbon electrodes, non-metals, organic conductors, semiconductor electrodes, diamond electrodes, etc. may be used. This method is characterized by high sensitivity detection of any substance with different oxidation-reduction potential in the medium by manipulating the electrode potential, and the miniaturization processing is extremely easy due to the simple structure of the electrode. Have.

  In addition, an electrode capable of detecting the membrane potential can be used when a change in membrane potential is caused by the permeation of the material into the membrane, the adhesion of the material to the membrane, adsorption, deposition, and interaction with the membrane. The membrane potential includes a Donnan potential, a diffusion potential, and a surface potential (interface potential). However, the membrane potential is not particularly limited as long as these potentials can be detected. Saturated calomel electrode, silver / silver chloride electrode, silver / silver ion electrode (eg Ag / 0.01M AgClO4 + supporting electrolyte), standard redox system {ferrocene / ferricinium (Fc / Fc +) system or bisphenylchrome (0) / ( 1) Electrodes of (BCr / BCr +)}, gold, platinum, carbon electrodes, platinum-carbon electrodes, tungsten, titanium, semiconductor electrodes (eg, titanium dioxide), etc. can be used.

  In addition, in order to detect ions and substances in the microreactor and detect the membrane potential, a microelectrode, a semiconductor electrode (eg, titanium dioxide), a semiconductor modified electrode, an n-type semiconductor electrode, an n-type coated with a conductive polymer. One or a plurality of electrodes can be disposed on a type semiconductor electrode, a p-type semiconductor electrode, a light-sensitive thin film, or a newly generated substance at the liquid / liquid interface.

  The above electrodes can be integrated with the material constituting the microchannel, or can be arranged separately.

  Further, it is desirable that the counter electrode or reference electrode (or salt bridge) of these detection microelectrodes be disposed on the same liquid phase side as the working electrode at a position that does not hinder the diffusion of substances from the molecular film. You may arrange | position to the separated liquid phase side.

  The reaction unit 14 causes various chemical reactions, physical and chemical phenomena to occur in the molecular film 5 and fluid in the microreactor 1 by light irradiation or the like.

  The circulation unit 15 collects the fluid used for the reaction in the microreactor 1 and circulates it to the first inlet 22, the second inlet 23, the third inlet 21, etc., thereby increasing the efficiency of the reaction. .

  The separation / recovery unit 16 recovers the product generated in the microreactor 1. For example, the product is recovered by a method such as ultrafiltration, chromatography or crystallization.

  FIG. 6 shows a specific example 1 of the microreactor analysis system. In this case, electrostatic, hydrophobic, hydrophilic and ionic interactions with the molecular film (lipid bilayer) of the substance (B) contained in the first fluid 26 or the second fluid 27, bioaffinity By observing changes in the electrochemical properties of the membrane, such as the current, potential, resistance, capacitance, and electrical signal pattern of the molecular membrane (lipid bilayer) caused by the first fluid 26 or the second Qualitative and quantitative measurement of the substance (B) or sample contained in the fluid 27 is performed. Alternatively, the molecular film (lipid) contained in the first fluid 26 or the second fluid 27 is applied by applying voltage or irradiating light to the molecular film (lipid bimolecular film) in the microreactor 1. Changes in electrostatic, hydrophobic, hydrophilic and ionic interactions with bilayers), changes in bioaffinity, or photo-sensitive substances, photo-sensitive molecules contained in molecular membranes (lipid bilayers) Membrane current, potential, resistance, capacitance, and electrical signal pattern caused by changes in the permeation of substances (B) such as ions and redox species to the molecular membrane (lipid bilayer membrane) caused by structural changes in the light channel A microreactor that performs qualitative and quantitative measurement of the substance (B) or sample contained in the first fluid 26 or the second fluid 27 by observing changes in the electrochemical characteristics of the film, such as changes in This is an analysis system (1) (biosensing system).

  FIG. 7 shows a specific example 2 of the microreactor analysis system. In this case, electrostatic, hydrophobic, hydrophilic and ionic interactions with the molecular film (lipid bilayer) of the substance (B) contained in the first fluid 26 or the second fluid 27, bioaffinity Is to detect the substance (B) by directly detecting changes in the absorption spectrum, fluorescence spectrum and heat of the fluid and molecular film in the microchannel. Alternatively, the permeation change of the substance (B) such as ions to the molecular membrane (lipid bilayer membrane) caused by the above interaction is measured by measuring the fluorescence or absorption spectrum of the substance (B) that has passed through the molecular membrane, Measurement of the thermal signal of the first fluid 26 or the second fluid 27, or the change in the fluorescence spectrum of the first fluid 26 or the second fluid 27 caused by the transmitted substance (B). 26 or the substance (B) or sample contained in the second fluid 27 is quantified or qualitatively determined.

  Alternatively, the molecular film (lipid) contained in the first fluid 26 or the second fluid 27 is applied by applying voltage or irradiating light to the molecular film (lipid bimolecular film) in the microreactor 1. Caused by electrostatic, hydrophobic, hydrophilic and ionic interactions with bilayers), changes in bioaffinity, or changes in the structure of light-sensitive substances, light-sensitive molecules or light channels in the molecular film Changes in the permeation of the substance (B) such as ions and redox species into the molecular membrane (lipid bilayer membrane), measurement of the fluorescence or absorption spectrum of the substance (B) that has passed through the molecular membrane, the first fluid 26 or The first fluid 2 can be measured by measuring the thermal signal of the second fluid 27 or by measuring the change in the fluorescence spectrum of the first fluid 26 and the second fluid 27 caused by the transmitted substance (B). Or a substance contained in the second fluid 27 (B) or the microreactor analysis system for quantitative or qualitative sample (2) (biosensing system).

  FIG. 8 shows a third specific example of the microreactor analysis system. In this case, electrostatic, hydrophobic, hydrophilic and ionic interactions with the molecular film (lipid bilayer) of the substance (B) contained in the first fluid 26 or the second fluid 27, bioaffinity By detecting the change in the permeation of the substance (B) such as ions and redox species into the molecular membrane (lipid bilayer membrane) caused by the first fluid, the first fluid Qualitative and quantitative measurement of the substance (B) or sample contained in 26 or the second fluid 27 is performed.

  Alternatively, the molecular film of the substance (B) contained in the first fluid 26 or the second fluid 27 (lipid bimolecular film) as the voltage is applied to the lipid bimolecular film in the microreactor 1 or light is irradiated. Molecular membranes caused by electrostatic, hydrophobic, hydrophilic and ionic interactions with, changes in bioaffinity, or changes in the structure of light sensitive substances, light sensitive molecules or light channels in molecular films ( The first fluid 26 or the second fluid 27 is detected by detecting the permeation change of the substance (B) such as ions and redox species into the lipid bilayer membrane) by microelectrodes installed inside and outside the microchannel. Is a microreactor analysis system (3) (biosensing system) that performs change qualitative and quantitative measurement of the substance (B) or sample contained in.

  In addition to the three types of microreactor analysis systems described above, electrostatic, hydrophobic, hydrophilic and ionic interactions with the molecular film of the substance (B) contained in the first fluid 26 or the second fluid 27 , Changes in the molecular film surface state caused by bioaffinity, optical microscope, electron microscope, laser electron microscope, differential interference phase contrast microscope, scanning electron microscope (SEM), Fourier transform infrared analyzer, laser Raman spectrometer, Analyzing the substance (2) or sample contained in the first fluid 26 and the second fluid 27 using a surface analyzer such as a fluorescence microscope, a polarization microscope, an X-ray analyzer and an atomic force microscope (AFM) There is a macro reactor analysis system (4) (biosensing system) capable of

(Method for producing molecular film)
Next, a method for producing a molecular film will be described.

  (A) First, as shown in FIG. 2, the first fluid 26 or the second fluid 27 is introduced from the first introduction port 22 and the second introduction port 23, and a stable liquid / A liquid laminar flow is formed.

  (B) Next, a fixed amount of the amphiphilic molecule mixed solution 25 is introduced from the third inlet 21 continuously, intermittently or for a fixed time.

  Alternatively, the amphiphilic molecule mixed solution 25, the first fluid 26 or the second fluid 27 is simultaneously introduced into the microchannel from the microchannel inlet.

  The flow rate and flow rate of the amphiphilic molecule mixed solution 25, the first fluid 26 or the second fluid 27 are the pressure of the feed pump, the temperature of the system, the amphiphilic molecule mixed solution 25, the first fluid 26 or the second fluid. This relates to various factors such as the viscosity derived from the composition of the fluid 27, the cross-sectional area of the microchannel, the material constituting the injection channel or the microchannel, the length and the three-dimensional shape, but in the first embodiment, it is stable. If a large area and a stationary or flowing molecular film can be formed, the amphiphilic molecule mixed solution 25, the first inlet 25, the first inlet 22, and the second inlet 23 are passed through the third inlet 21, the first inlet 22, and the second inlet 23. The various conditions for introducing the second fluid 26 or the second fluid 27 are not limited.

  The microchannel 3 may have a groove shape formed in the microreactor 1 as shown in FIG. 2, or may have a ring shape as shown in FIG. In addition, it may have a polygonal shape such as an elliptical shape, a rectangular shape, or a hexagonal shape, depending on conditions such as the use, the type of amphiphilic molecule (lipid), and the constituent components of the molecular film. Further, depending on the shape of the microchannel 3, the molecular film to be produced has a planar shape or a cylindrical shape. FIG. 2B is a schematic diagram of a planar bimolecular film, and FIG. 4B is a schematic diagram of a cylindrical bimolecular film 35.

  Further, by integrating the single-tube microchannels shown in FIG. 5A, a multi-tube microchannel shown in FIG. 5B can be provided. In the first embodiment, the system scale-up method is not particularly limited as long as it has a microreactor function having a similar molecular film.

  In order to form a laminar flow in the microchannel, the Reynolds number Re must be Re ≦ 2100. On the other hand, Re has the following relationship with the microchannel diameter (D), flow velocity (ν), density (ρ), and solution viscosity (μ).

Re = Dνρ / μ
Therefore, in the present invention, when a laminar flow is obtained by appropriately combining the diameter, flow rate, density and solution viscosity of the microchannel, the type of fluid, the material of the channel substrate, the type of liquid feed pump, the channel diameter It is not limited to conditions such as size.

  In order to form a stable lipid bilayer, it is desirable that the linear velocity (L) of each laminar flow in the microchannel be the same. The linear velocity has the following relationship with the fluid density (ρ) and the cross-sectional area (S) of the tube.

ρ1 × L1 × S1 = ρ2 × L2 × S2 (1)
When the linear velocity (L) is the same, that is, when L1 = L2, the equation (1)
ρ1 × S1 = ρ2 × S2 (2)
Is obtained. That is, the density of the fluid and the cross-sectional area of the tube have a relationship as shown in the equation (3).

S1 / S2 = ρ2 / ρ1 (3)
As is clear from the above relational expression, when fluids having different densities are flowed in order to obtain a constant linear velocity, this can be realized by adjusting the cross-sectional area of the tube.

  (C) The amphiphilic molecule mixed liquid 25 introduced from the third inlet 21 is introduced between the liquid / liquid laminar flow, between the gas / liquid laminar flow, or between the liquid laminar flow / wall surface. Then, in the liquid / liquid laminar flow, the gas / liquid laminar flow, or the liquid laminar flow / wall surface, the solvent for dissolving the amphiphilic molecule mixed solution 25 is gradually diffused to the wall surface of each laminar flow phase or channel. Thus, a stationary molecular film having a stable two-dimensional or three-dimensional area can be produced between the liquid / liquid laminar flow, the gas / liquid laminar flow, or the liquid laminar flow / wall surface.

  Next, a method for manufacturing an integrated film (molecular integrated film) will be described with reference to FIGS.

  As shown in FIG. 9A, first, an amphiphilic molecule mixed solution is introduced from the third introduction port 21, and a fluid is introduced from the first introduction port 22. The third inlet 21 and the first inlet 22 are movable, and a movable barrier 40b is also installed at the outlet. When the lipid bilayer membrane (molecular membrane) 5 of the first layer is produced, the barrier 40a integrated with the third inlet 21 and the barrier 40b provided at the outlet are moved downward as shown in FIG. And the integrated film (molecular integrated film) is gradually formed with the second layer, the third layer,... At this time, the barrier 40c provided below the first inlet 22 may also be gradually moved upward. In addition, as the integrated film 7 increases, the channel through which the fluid flows becomes narrower and the flow velocity tends to increase, but this gradually decreases the inflow velocity of the fluid according to the width and size of the channel. Can be controlled. The pressure applied to the first introduction port 22 and the third introduction port 21 is P1> P2.

  Further, as shown in FIG. 10, the molecular film 5 is formed by sucking the barriers 40d, 40e, 40f provided at the outlet of the third introduction port 21 in the direction in which the amphiphilic molecule mixed solution is introduced. You may make it form.

  The molecular film 5 according to the first embodiment has various molecular film production conditions such as the type and composition of the amphiphilic molecule mixed solution 25, the first fluid 26 and the second fluid 27, and the outflow speed. Depending on the structure of monolayer, bilayer, lipid bilayer, reverse bilayer, lipid multilayer, molecular multilayer, micelle, reverse micelle, vesicle, reverse vesicle, emulsion and LB membrane alone Alternatively, it can exist in a mixed form.

  According to the method for producing a molecular film according to the first embodiment, since both liquid phases in the microchannel are laminar, a stable molecular film or a lipid bimolecular film can be produced. Further, a bilayer membrane or lipid bilayer membrane having a large area can be produced by extending the length of the channel. In addition, rapid diffusion of a solvent that dissolves amphiphilic molecules or lipids enables rapid production of bilayers or lipid bilayers. Furthermore, it becomes easy to regenerate the deteriorated film.

(Characteristics and application of microreactor with molecular film)
Typical reaction engineering characteristics of a microreactor equipped with a molecular film include the following points.

1) No shearing of the enzyme occurs with stirring.

2) A complicated enzyme immobilization treatment is unnecessary, and there is no degradation of enzyme activity due to the immobilization treatment.

3) Almost no side reaction occurs.

4) The reaction temperature can be easily controlled and the reaction rate can be controlled.

5) Since the concentration distribution hardly occurs, the reaction system can be easily scaled up.

6) Since the specific surface area and specific interface area are large, the molecular diffusion distance is short, and the diffusion-controlled reaction is dramatically accelerated.

7) Since an agitation power for causing forced convection is not required, an energy-saving reaction system can be constructed.

8) Since the reaction heat can be diffused quickly, a rapid reaction can be caused.

  Features of the microreactor including the molecular film according to the first embodiment of the present invention include the following points.

1) In application to a bioreactor or a biosensor, a complicated enzyme immobilization treatment is unnecessary, and further, there is no degradation of enzyme activity due to the immobilization treatment.

2) It is easy to regenerate the deteriorated film.

3) A microreactor having a lipid bilayer membrane has all the functions of a biological membrane.

  For example, the lipid bilayer membrane in the microchannel is fluid as well as the advantage of fluidity in the biological membrane. The following advantages can be mentioned depending on the fluidity of the membrane.

1) The molecular metabolic exchange takes place continuously and the membrane can adapt to the new environment as well as the cells.

2) The protein can move in the membrane.

The lipid bilayer membrane in the microchannel has the same characteristics as the biological membrane. For example,
1) The functional expression of membrane protein molecules and this complex is closely related to membrane lipids.

  2) The membrane has fluidity depending on the fatty acid composition and the phospholipid / cholesterol ratio.

  3) The membrane has a front and back.

The lipid bilayer membrane in the microchannel can perform the same function as the biological membrane. For example,
(1) It becomes a boundary between both liquid phases.

  (2) Transporting substances.

  (3) It will be a place to convert materials and energy.

  (4) Accept, transmit and process information.

  To summarize the functions of the above biological membranes, the lipid bilayer membrane, which is a biological membrane, is not only a partition that isolates the cell membrane from the outside world and other cells, but also substance recognition and permeation, information conversion and transmission, enzyme reaction, etc. It plays an important role as a place. Similar to the function of the biological membrane, the present invention can create an artificial cell membrane model having various biological functions or new functions, and the function can be expressed and realized by the microreactor.

(Micro bioreactor with molecular membrane)
Specifically, using the characteristics of the biological membrane function as described above, for example, if a microreactor system having a molecular membrane such as a biological membrane is applied to a bioreactor process, a reaction rate of 100% is achieved in the enzyme reaction system. Can be achieved. This bioreactor process will be described with reference to FIGS.

  The lipid mixture shown in FIGS. 11 to 19 is a case where lipid is used as the amphiphilic molecule when preparing the amphiphilic molecule mixture. The amphiphilic molecules that form the molecular membrane of the microbioreactor including the molecular membrane are not particularly limited to lipids.

  In addition, in FIGS. 11 to 19, the reaction system of the microbioreactor provided with the molecular film is a soaking system {water phase (W) / water phase (W)} and {oil phase (O) / oil phase (O)}. Alternatively, examples of the heterophasic system {oil phase (O) / water phase (W)} will be listed to describe the process of the microbioreactor. In the case of the heterophasic system, {water phase (W) / gas phase (V )} Or a combination of {oil phase (O) / gas phase (V)}.

  For example, as shown in FIG. 11, the microreactor 1 includes an amphiphilic molecule mixed solution (lipid mixed solution 25) (L) from the third inlet 21 and a substrate (S) from the first inlet 22. Solution, solution containing enzyme + substrate (E + S) or enzyme (E) is introduced from second inlet 23, respectively, soaking system {aqueous phase (W) / aqueous phase (W)} or heterophasic system {oil Phase (O) / water phase (W)}. The substrate (S) passes through the molecular film 5, forms a complex with the enzyme (E), and a product (P) is generated. The product (P) can permeate the molecular film 5. An antibiotic is mentioned as a product produced | generated in this way. For example, in the process of FIG. 11, as shown in FIG. 12, 7-aminocephalosporanic acid is produced. Further, when the product (P) is recovered, the unreacted substrate (S) is returned to the first inlet 22. The enzyme in this process is immobilized in a certain space of the microchannel.

  According to the bioreactor process shown in FIG. 11, antibiotics and the like can be produced.

  In addition, as shown in FIG. 13, in the microreactor 1, a liquid mixture (L) containing the enzyme (E) from the third inlet 21 and a solution containing the substrate (S) from the first inlet 22 {water Phase (W) or oil phase (O)}, and the solution {water phase (W) or oil phase (O)} is introduced from the second inlet 23, respectively, and the soaking system {water phase (W) / water phase (W)} or {oil phase (O) / oil phase (O)}. The substrate (S) forms a complex with the enzyme (E) in the molecular film 5 to produce a product (P). The enzyme in this process is immobilized on the molecular membrane. A steroid is mentioned as a product produced | generated in this way. For example, in the process of FIG. 13, a steroid is generated as shown in FIG. Further, when the product (P) is recovered, the unreacted substrate (S) is returned to the first inlet 22.

  According to the bioreactor process shown in FIG. 13, steroids and the like can be produced. In the case of a diffusion system reaction, the problem of substrate or product diffusion rate control can be solved by miniaturizing the reaction system.

  As shown in FIG. 15, the bioreactor process described in FIG. 11 collects the product (P) that has not passed through the molecular membrane 5 and the product (P) that permeates the molecular membrane, You may further provide the structure which recycles (E) and an unreacted substrate (S). FIG. 15 is similar to FIG. 11, in the microreactor 1, an aqueous phase (W) or an oil phase (L) containing a lipid mixture (L) from the third inlet 21 and a substrate (S) from the first inlet 22. O), the aqueous phase (W) containing the enzyme + substrate (E + S) is introduced from the second inlet 23, and the homogeneous phase system {aqueous phase (W) / aqueous phase (W)} or heterogeneous system {oil phase (O) / water phase (W)}. The substrate (S) passes through the molecular film 5, forms a complex with the enzyme (E), and a product (P) is generated. At this time, the product (P) that has not passed through the molecular membrane 5 is recovered by a method such as ultrafiltration, chromatography, or crystallization, and the enzyme (E) and unreacted substrate (S) are secondly recovered. Return to the inlet 23. In addition, the product (P) that permeates the molecular membrane is separated by methods such as ultrafiltration, chromatography and crystallization, and the product (P) is recovered and the unreacted substrate (S) is first introduced. Return to mouth 22 or second inlet 23.

  According to the bioreactor process shown in FIG. 15, the product (P), enzyme (E), and unreacted substrate (S) can be quickly recovered.

  Also, as shown in FIG. 16, the bioreactor process described in FIG. 13 is a method such as ultrafiltration, chromatography, crystallization, etc., for the product (P) produced in the microchannels on both sides of the molecular membrane 5. And a structure for recycling the unreacted substrate (S). FIG. 16 is similar to FIG. 13, the lipid mixture (L) containing the enzyme (E) from the third inlet 21, the water phase (W) or the oil phase (O) from the second inlet 23, the first The water phase (W) or the oil phase (O) containing the substrate (S) is introduced from the introduction port 22 of the water, respectively, and the soaking system {water phase (W) / water phase (W)} or {oil phase (O) / Oil phase (O)}. The substrate (S) forms a complex with the enzyme (E) in the molecular film 5 to produce a product (P). At this time, the product (P) produced on both sides of the molecular film 5 is recovered by a method such as ultrafiltration, chromatography or crystallization, and unreacted substrate (S) is removed from the second inlet and the second inlet. 2 to the inlet 23.

  According to the bioreactor process shown in FIG. 16, the product (P) and the unreacted substrate (S) can be quickly recovered.

  In addition, as shown in FIG. 17, in the microreactor 1, a solution containing the lipid mixture (L) from the third inlet 21, a solution containing the substrate (S 2) from the first inlet 22, and from the second inlet 23. A solution containing the enzyme + substrate (E + S1) is introduced to obtain a soaking system {aqueous phase (W) / aqueous phase (W)} or a heterogeneous system (oil phase (O) / aqueous phase (W)). The substrate (S2) passes through the molecular film 5, forms a complex with the enzyme (E) together with the substrate (S1), and a product (P1) and a product (P2) are generated. Only the product (P2) can permeate the molecular film 5. Then, when recovering the product (P1), the enzyme (E) and the unreacted substrates (S1, S2) are returned to the second inlet 23. On the other hand, when the product (P2) is recovered, the unreacted substrate (S2) is returned to the first inlet 22.

  According to the bioreactor process shown in FIG. 17, separation of products (P1, P2) and rapid recovery of unreacted substrates (S1, S2) are possible.

  In addition, as shown in FIG. 18, in the microreactor 1, a solution containing the lipid (L) containing the enzyme (E) from the third inlet 21 and the substrate (S2) from the first inlet 22, The solution containing the substrate (S1) is introduced from each of the two inlets 23 to obtain a heterophasic system {oil phase (O) / water phase (W)}. The substrates (S1, S2) form a complex with the enzyme (E) in the molecular film 5, and products (P1, P2) are generated. Then, when the product (P1) is recovered, the unreacted substrate (S1) is returned to the second inlet 23. On the other hand, when the product (P2) is recovered, the unreacted substrate (S2) is returned to the first inlet 22.

  According to the bioreactor process shown in FIG. 18, separation of products (P1, P2) and rapid recovery of unreacted substrates (S1, S2) are possible.

  Further, as shown in FIG. 19, in the microreactor 1, a lipid mixture (L) containing the enzyme (E) from the third inlet 21, a solution containing the substrate (S 2) from the first inlet 22, The solution containing the substrate (S1) is respectively introduced from the inlet 23 of No. 2 to obtain a soaking system {aqueous phase (W) / aqueous phase (W)} or {oil phase (O) / oil phase (O)}. . The substrates (S1, S2) form a complex with the enzyme (E) in the molecular film 5, and products (P1, P2) are generated. Then, when the product (P1) is recovered, the unreacted substrate (S1) is returned to the second inlet 23. On the other hand, when the product (P2) is recovered, the unreacted substrate (S2) is returned to the third inlet 21.

  According to the bioreactor process shown in FIG. 19, separation of products (P1, P2) and rapid recovery of unreacted substrates (S1, S2) are possible.

(Membrane fusion and extraction by microreactor with molecular membrane)
Further, in the first embodiment, as shown in FIG. 20, bio-related substances 9 such as proteins and nucleic acids in cells or viruses having lipid bilayers are extracted by membrane fusion with lipid bilayers 5. can do. When this method is used, it is not necessary to go through a series of extraction and purification processes such as cell crushing in order to detect viruses, and the causative protein and the nucleic acid of the virus can be directly extracted.

  Further, in the first embodiment, in FIG. 20, the electrodes 51 a and 51 b installed on both sides of the film by the voltage applying device which is one of the condition control units 12 are instantaneously applied with a high direct current voltage or alternating current. Apply a voltage of. At this time, since the lipid bilayer membrane 5 is disturbed, cell fusion is likely to occur. When the cell 8 is fused with the lipid bilayer membrane 5, the cell organelles (nucleic acid, protein, endoplasmic reticulum, Golgi apparatus, ribosome, etc.) are released in the same manner as in vivo cell fusion. Moreover, by using together with other detection methods (for example, a protein sensor and a gene sensor), the causative protein and the nucleic acid extracted with high sensitivity, simple and rapid can be detected. The stimulus applied to the molecular film is not limited to the electrical stimulus described above, and the same effect as the electrical stimulus can be obtained by changing the pH of the fluid, the temperature of the fluid and the molecular film, and the like.

  In addition, as shown in FIG. 21, by applying a voltage to the lipid bilayer membrane 5, the biological material 9, particularly protein, in the fluid is extracted to the liquid phase on the opposite side of the lipid bilayer membrane 5. As the electrodes 51a and 51b, it is possible to use microelectrodes having an electrode surface such as a line shape or a dot shape. Peptides, polypeptides, proteins and the like synthesized by cell-free protein synthesis can be extracted by the same extraction method. In this case, since the produced protein is immediately extracted and separated, protein aggregation can be prevented.

(Operation and effect of the microreactor according to the first embodiment)
According to the microreactor according to the first embodiment, the monolayer, the bilayer, the lipid bilayer, the reverse bilayer, which can be retained or flowed, have a stable large area, and can introduce various substances. Molecular film, molecular multilayer film, lipid multilayer film, micelle, reverse micelle, vesicle, reverse vesicle, emulsion, LB film, or a mixture of these, two-dimensional planar, three-dimensional columnar A molecular film having at least one of a conical shape, a truncated conical shape, a cylindrical shape, an elliptical cylindrical shape, and a polygonal shape can be produced automatically, rapidly, and continuously. Further, by using the microreactor according to the first embodiment, various rapid chemical reactions, bioreactors, separation (extraction) systems, and biosensing systems can be provided.

  In addition, according to the microreactor according to the first embodiment, a lipid bimolecular membrane having the same function as that of a biological membrane can be produced and provided as a new microchemical reaction field, and various information transmission and substances in the biological membrane can be provided. It can contribute to the elucidation of the permeation mechanism.

  In addition, according to the microreactor according to the first embodiment, it is possible to confirm and predict the influence of a new chemical substance or a new drug on a biological system. Furthermore, it is possible to provide a novel trace amount / high sensitivity biosensing, a high efficiency (100%) bioreactor (normal temperature, normal pressure) and a high efficiency, continuous, low byproduct rapid chemical reaction system.

  Examples according to the first embodiment of the present invention will be described below.

(Comparative Example 1)
As shown in FIG. 22, 3 ml of 50 mM Tris buffer (pH 8.5) containing 3 M ethanol and 0.5 mM NAD + is added as the first fluid 26 to a quartz cell adjusted to a constant temperature of 30 ° C. The reaction is started by adding 100 μl of 50 mM Tris buffer (pH 8.5) containing 30 μg / l alcohol dehydrogenase as the second fluid 27.

  The progress of the reaction is followed by absorbance at 340 nm (NADH's characteristic absorption wavelength).

  The reaction of Comparative Example 1 is a reaction when no molecular film is present in the microrechannel.

Example 1
As shown in FIG. 22, an n-decane solution containing 5 mg / ml lecithin as an amphiphilic molecule mixed solution (lipid mixed solution 25) in a homogeneous phase system {aqueous phase (W) / aqueous phase (W)} First fluid 26 contains 50 mM Tris buffer (pH 8.5) containing 0.3 M ethanol and 0.1% bovine serum albumin, and second fluid 27 contains 0.5 mM NAD +, 1 μg / l alcohol dehydrogenase and 0.1% bovine serum albumin. Use 50 mM Tris buffer (pH 8.5). Other conditions and products are as follows.

  The reaction of Example 1 is a reaction when a molecular film is present in the microchannel.

Enzyme: Alcohol dehydrogenase Substrate: Ethanol coenzyme: Nicotinamide adenine dinucleotide (oxidized form) (NAD +)
Products: acetaldehyde, nicotinamide adenine dinucleotide (reduced form) (NADH)
The first fluid 26 is caused to flow at a predetermined flow rate from the first inlet 22 of the microreactor adjusted to a constant temperature condition of 30 ° C. On the other hand, the second fluid 27 is caused to flow from the second inlet 23 at the same flow rate as that of the first inlet 22. Further, an amphiphilic molecule mixed solution (lipid mixed solution 25) is caused to flow from the third inlet 21 at a predetermined flow rate. The following reaction occurs in the microreactor 1.

CH3CH2OH + NAD + = CH3CHO + NADH + H +
The progress of the reaction is followed by absorbance at 340 nm (NADH's characteristic absorption wavelength).

(result)
Compared with the system of Comparative Example 1, the system of Example 1 improved both the reaction rate and the conversion of acetaldehyde at a constant reaction time. Specifically, the reaction rate was improved about 4 times, and the conversion rate of acetaldehyde after 5 minutes was improved about 3 times (see FIG. 23).

  The characteristics of the high reaction rate and high conversion shown in the enzyme reaction system using the microreactor including the molecular film according to the first embodiment are considered to be due to the following two typical characteristics. .

  (1) Since ethanol, which is a substrate, also acts as an enzyme denaturant in the solution, if there is no lipid bilayer, the enzyme loses its activity stability when the ethanol concentration increases. In the case where there is a lipid bilayer membrane, ethanol gradually diffuses through the lipid bilayer membrane into the aqueous phase where the enzyme is present, so that such denaturation of the enzyme can be avoided. Further, in the conventional system, in order not to lose the activity of the enzyme, the reaction must be carried out in a solution containing a low concentration of ethanol, so that only acetaldehyde which is a low concentration reaction product can be obtained. In order to obtain a high concentration of acetaldehyde, there is a further problem that it must be concentrated using another means. By using this system, the above problems can be solved, and production of a high concentration of acetaldehyde can be achieved.

  (2) The chemical equilibrium is shifted, and the conversion rate and reaction yield can be increased. This enzyme reaction system is a reversible system, and when there is no lipid bilayer membrane, the production of the product acetaldehyde has a problem that the reaction stops when chemical equilibrium is reached, and a high reaction yield is obtained. I can't. By using a microreactor equipped with a molecular membrane, the acetaldehyde produced by the enzyme reaction is quickly diffused to the opposite side of the lipid bilayer membrane and excluded from the enzyme reaction side, so that the chemical equilibrium is in the reaction direction to produce acetaldehyde. Shift and high reaction yield can be obtained.

(Example 2)
As shown in FIG. 24, in the heterophasic system {oil phase (O) / water phase (W)}, the n-decane solution containing 5 mg / ml diolein as the amphiphilic molecule mixture (lipid mixture 25), As a first fluid 26, a chloroform solution containing 0.1 mM N-acetyl-L-tryptophan and 10 mM ethanol, and as a second fluid 27, a 0.1 M phosphate buffer (pH 6.8) containing 0.5 g / l α-chymotrypsin. Use. Other conditions and products are as follows.

Enzyme: α-chymotrypsin substrate: N-acetyl-L-tryptophan (AT-OH), ethanol (EtOH)
Product: N-acetyl-L-tryptophan ethyl ester (AT-OEt), water
The first fluid 26 is caused to flow at a predetermined flow rate from the first inlet 22 of the microreactor adjusted to a constant temperature condition of 30 ° C. On the other hand, the second fluid 27 is caused to flow from the second inlet 23 at the same flow rate as that of the first inlet 22. Further, an amphiphilic molecule mixed solution (lipid mixed solution 25) is caused to flow from the third inlet 21 at a predetermined flow rate. The following reaction occurs in the microreactor 1.

AT-OH + EtOH = AT-OEt + H2O
(result)
The reaction product N-acetyl-L-tryptophan ethyl ester was detected and quantified by high performance liquid chromatography.

  The measurement conditions for high performance liquid chromatography are as follows.

  ODS (0.15 mX6.0 mmφ) was used for the column, and a mixed solution of pure water and acetonitrile (1: 1 by volume) was flowed at a flow rate of 1 ml / min-1 as a fluid phase. An ultraviolet absorption detector (wavelength 270 nm) was used.

  FIG. 25 shows the relationship between the yield of N-acetyl-L-tryptophan ethyl ester and the flow rate ratio. As shown in FIG. 25, the yield of N-acetyl-L-tryptophan ethyl ester decreased as the flow rate ratio increased. That is, in Example 2, the production amount per unit time can be directly and freely controlled by changing the flow rate ratio. Furthermore, the formation of the monomolecular film can prevent the deterioration of activity due to the contact of the enzyme at the chloroform and water interface.

(Example 3)
As shown in FIG. 26, in the soaking system {oil phase (O) / oil phase (O)}, 5 mg / ml subtilisin Carlsberg, a small amount of water and 5 as an amphiphilic molecule mixture (lipid mixture 25) A hexane solution containing mg / ml lecithin, a hexane solution containing 200 mM S-α-methylbenzyl alcohol as the first fluid 26, and a hexane solution containing 200 mM vinyl butanoate as the second fluid 27 are used. Other conditions and products are as follows.

Enzyme: Subtilisin Carlsberg
Substrate: S-α-methylbenzyl alcohol {C6H5CH (CH3) OH}
Vinyl butanoate (CH3CH2CH2COOCH = CH2)
Products: butanoic acid α-methylbenzyl ester (CH3CH2CH2COOCH (CH3) C6H5),
Vinyl alcohol (CH2 = CHOH)
The first fluid 26 is caused to flow at a predetermined flow rate from the first inlet 22 of the microreactor adjusted to a constant temperature condition of 30 ° C. On the other hand, the second fluid 27 is caused to flow from the second inlet 23 at the same flow rate as that of the first inlet 22. Further, an amphiphilic molecule mixed solution (lipid mixed solution 25) is caused to flow from the third inlet 21 at a predetermined flow rate. The following reaction occurs in the microreactor 1.

C6H5CH (CH3) OH + CH3CH2CH2COOCH = CH2
= CH3CH2CH2COOCH (CH3) C6H5 + CH2 = CHOH
(result)
The reaction product, butanoic acid α-methylbenzyl ester, was detected and quantified by gas chromatography.

  The measurement conditions for gas chromatography are as follows.

  A Chirasil-DEX CB capillary column (Chrompack) was used as the column, and nitrogen was flowed at 0.5 ml / min as the carrier gas. A flame ionization detector (FID) was used.

Example 4
In a homogeneous phase system {aqueous phase (W) / aqueous phase (W)} having the same configuration as in FIG. 22 (Example 1), 5 mg / ml monoolein was used as the amphiphilic molecule mixture (lipid mixture 25). N-decane solution containing, 50 mM Tris buffer (pH 8.5) containing 0.1 M glutaryl 7-aminocephalosporanic acid and 0.1 g / l GL7-ACA amidase as first fluid 26, as second fluid 27 Use 50 mM Tris buffer (pH 8.5). Other conditions and products are as follows.

Enzyme: GL7-ACA amidase (Pseudomonas sp. GK-16 origin)
Substrate: glutaryl 7-aminocephalosporanic acid (glutaryl 7-ACA)
Product: 7-aminocephalosporanic acid (7-ACA)
The first fluid 26 is allowed to flow at a predetermined flow rate from the first inlet 22 of the microreactor adjusted to a constant temperature condition of 20 ° C. On the other hand, the second fluid 27 is caused to flow from the second inlet 23 at the same flow rate as that of the first inlet 22. Further, an amphiphilic molecule mixed solution (lipid mixed solution 25) is caused to flow from the third inlet 21 at a predetermined flow rate. In the microreactor 1, the reaction of Example 4 shown in FIG.

(result)
The reaction product 7-aminocephalosporanic acid was detected and quantified by high performance liquid chromatography.

  The measurement conditions for high performance liquid chromatography are as follows.

  ODS (0.15 mX6.0 mmφ) was used for the column, and a mixed solution of pure water and acetonitrile (1: 1 by volume) was flowed at a flow rate of 1 ml / min-1 as a fluid phase. A differential refraction detector was used.

(Example 5)
In a phase-homogeneous system (aqueous phase (W) / aqueous phase (W)) having the same structure as in FIG. 22 (Example 1), 5 mg / ml monoolein was used as the amphiphilic molecule mixture (lipid mixture 25). N-decane solution containing, 10 mg / ml Arthrobacter simplex acetone-dried cells as the first fluid 26, and 20 mM phosphate buffer (pH 7.0) containing 0.5 g / l hydrocortisone and 20% ethanol as the second fluid 27 ) Is used. Other conditions and products are as follows.

Cell: Acetone-dried cell of Arthrobacter simplex Substrate: Hydrocortisone Product: Prednisolone
The first fluid 26 is caused to flow at a predetermined flow rate from the first inlet 22 of the microreactor adjusted to a constant temperature condition of 30 ° C. On the other hand, the second fluid 27 is caused to flow from the second inlet 23 at the same flow rate as that of the first inlet 22. Further, an amphiphilic molecule mixed solution (lipid mixed solution 25) is caused to flow from the third inlet 21 at a predetermined flow rate. In the microreactor 1, the reaction of Example 5 shown in FIG. 14 occurs.

(result)
The reaction product prednisolone was detected and quantified by high performance liquid chromatography.

  The measurement conditions for high performance liquid chromatography are as follows.

  ODS (0.15 mX6.0 mmφ) was used for the column, and a mixed solution of pure water and acetonitrile (1: 1 by volume) was flowed as a fluid phase at a flow rate of 1.5 ml / min-1. Further, an ultraviolet absorption detector (wavelength 230 nm) was used.

(Example 6)
FIG. 27 shows an example of a sensing method for sensing light using a microreactor having a molecular film. As shown in FIG. 27, decane containing 5% azobenzene (p-Benzoquinone) / monoolein (1:10) as an amphiphilic molecule mixture (lipid mixture 25) (add chloroform until azobenzene is dissolved). The solution, pure water as the first fluid 26, and 0.1M benzoquinone (Q) solution as the second fluid 27 were used. The lipid bilayer membrane 5 contains a photosensitive molecule azobenzene.

  The lipid bilayer was irradiated with UV light (laser) (wavelength: 355 nm) intermittently at intervals of 20 seconds every 0.5 seconds.

  Then, benzoquinone that permeated through the lipid bilayer membrane was detected by light irradiation with a fine platinum disk electrode (diameter = 10 μm) 73 installed in the channel on the opposite side of the membrane. Specifically, a constant potential of -500 mV was applied to a small platinum disk electrode with respect to a silver / silver chloride electrode, and a supersensitive potentiostat (EG & G PARC type 283) was used with a three-electrode system, and benzoquinone The reduction current generated by the reduction reaction to hydroquinone was measured.

(result)
When the lipid bilayer was irradiated with light, a change in the reduction current of benzoquinone was observed with a small platinum disk electrode. On the other hand, when no light was irradiated, no change in the reduction current of benzoquinone was observed.

  This is because azobenzene contained in the lipid bilayer is a photosensitive molecule, and irradiating the membrane with UV light (355 nm) causes the photoisomerization reaction of azobenzene from the trans form to the cis form. Since the disorder of the lipid bilayer structure caused by this photoisomerization reaction causes a change in membrane permeation of benzoquinone, a change in reduction current caused by benzoquinone was detected on the opposite side of the membrane.

(Second Embodiment)
As shown in FIG. 28, the microchannel sensor according to the second embodiment has a microchannel in which a pair of electrodes or an electrode system having a constant interval is arranged in the microchannel along the direction of fluid flow. Fluid 61 (W), insulating medium 62 (O), fluid 63 (W), amphiphilic molecule mixture (M), fluid 65 (W) or fluid 65 (S) containing substance, insulation into channel Flowing in the order of the active medium 66 (O) and the fluid 67 (W).

  FIG. 28A shows the fluids 63 (W) and 65 (W), or the fluids 63 (W) and the fluid 65 layer containing the substance, with the amphiphilic molecule mixture 64 separated, and the electrodes 72 a and 72 b. It is the figure which reached below. Membrane electrical signals (eg, membrane potential, resistance, current, dielectric constant, capacitance, etc.) caused by the interaction of molecular membranes with substances contained in fluids, changes in membrane surface conditions, and fluids Quantification and qualification of a substance in a fluid is performed by measuring changes in the absorption spectrum and concentration of the substance.

  FIG. 28B is a diagram in which the fluid 65 (W) or the fluid 65 (S) containing a substance reaches below the sensor electrode 73, the reference electrode 74, and the counter electrode 75. Similarly, FIG. 28C is a diagram in which the fluid 63 (W) reaches under the sensor electrode 73, the reference electrode 74, and the counter electrode 75.

  As shown in FIG. 29, since the insulating medium 62 (O) or the insulating medium 66 (O) functions as a boundary wall of the electric field cell, the insulating medium 62 (O) in the microchannel. , Fluid 63 (W), amphiphilic molecule mixture 64 (M), fluid 65 (W) or fluid 65 (S) containing substance, and insulating medium 66 (O) function as one micro electric field cell. can do. Therefore, when a photosensitive molecule or substance is contained in the molecular film, electrons or holes are generated by photoexcitation of the photosensitive molecule or substance in the molecular film by light irradiation. These electrons or holes can be detected by an electrode system arranged to oxidize or reduce the electrochemical redox species in the fluid 63 or the fluid 65 and to oxidize or reduce the substance to be oxidized or reduced.

  Similarly to FIG. 29, as shown in FIG. 30, the insulating medium 62 (O) or the insulating medium 66 (O) has a function as a boundary wall of the electric field cell. The medium 62 (O), the fluid 63 (W), the amphiphilic molecule mixture 64 (M), the fluid 65 (W) or the fluid 65 (S) containing the substance, and the insulating medium 66 (O) Can function as two micro electric field cells. In FIG. 30F, for example, a molecular film containing an ion channel is used as a diaphragm, and an electrochemical reaction can be caused by the fluid 63 and the fluid 65. In FIG. 30 (g), for example, by installing salt bridges 77 connected to fluids on both sides of the molecular film, an electrochemical reaction using the molecular film as a diaphragm can be caused by the fluid 63 and the fluid 65.

  As shown in FIG. 31, the substance can be detected by examining the change in the surface state of the molecular film caused by the interaction between the substance (virus, amyloid protein, etc.) contained in the fluid 65 and the molecular film. . As a measuring method, incident light is introduced into the molecular film from one side of the channel, and the surface state of the film can be examined with light reflected from the film by an apparatus such as an optical microscope or a differential interference phase contrast microscope.

  In the case of FIG. 32 (c), the change in permeability of ions, hydrophobic substances, etc. caused by the interaction of substances (hydrophobic substances such as viruses and organic substances) contained in the fluid 65 with the molecular film is shown in the absorption spectrum. The target substance is detected by observing it as a change. In addition, due to the interaction (membrane fusion) of substances (hydrophobic substances such as viruses and organic substances) contained in the fluid 65, small organelles (nucleic acids, proteins, etc.) such as RNA, DNA and enzymes in viruses. Etc.), and the corresponding substance is detected by measuring the absorption spectrum of the released substance.

  The absorption spectrum measurement of FIG. 32 (c) is performed with the fluid 63. Similarly to FIG. 32 (c), when something released or generated by the interaction with the molecular film is present in the fluid 65, Measurement with the fluid 65 may be performed as shown in FIG.

  In the case of FIG. 33 (e), permeation of ions and the like caused by the interaction of a substance (virus, amyloid protein, etc.) contained in the fluid 65 with the molecular film, and the fluorescent agent present in the fluid 63 with the permeated ions. The substance is detected by detecting the fluorescence emitted by the interaction.

  In the case of FIG. 33 (f), RNA, DNA and enzyme in the virus, or organelle in the cell due to the interaction (for example, membrane fusion) of the substance (virus, cell, etc.) contained in the fluid 65 with the molecular film. (Nucleic acid, protein, etc.) is released, and the substance is detected by measuring the fluorescence emitted by the interaction with the fluorescent agent present in the fluid 63.

  The fluorescence measurements of FIGS. 33 (e) and (f) are performed with the fluid 63. However, as in FIGS. 33 (e) and (f), the fluid released or generated by the interaction with the molecular film is the fluid. When fluorescence is emitted by interaction with the fluorescent agent existing in 65, fluorescence measurement may be performed on the fluid 65 side.

  As shown in FIG. 34, qualitatively and quantitatively determine ions, proteins, nucleic acids, amino acids, organic substances, etc. by selectively collecting each segment such as fluid 63 or fluid 65 from the outlet and appropriately using them together with various analysis methods. To do. Various analysis methods include, for example, measurement of pH, viscosity, density, refractive index, fluorescence spectrum, absorption spectrum, measurement by HPLC, GC, GC / MS, FTIR, electrochemical measurement, etc. The analysis method related to the form may be the same as the analysis method of the first embodiment.

(Features of the microchannel sensor according to the second embodiment)
The fluid in the microchannel, the amphiphilic molecule mixture, and the insulating medium stay or flow in the microchannel in order to maintain the stable state of the formed molecular film.

  In addition, the insulating medium does not transmit ions, has a low dielectric constant, and has an effect of cleaning the contamination of the electrode caused by the fluid, the fluid containing the substance, and the molecular film. Examples of the insulating medium include a hydrophobic organic solvent (eg, hexane, decane, isooctane, octane, hexadecane), silicon oil, and fluorinated oil (eg, a mixture of dodecafluorotetradecane and tridecafluorooctanol (10: 1)). However, it is not particularly limited as long as it has an effect as an insulating medium. In addition, it is preferable that this droplet has an electrical insulating effect and an effect of cleaning the electrode.

  Further, a plurality of pairs of electrodes having a constant interval may be arranged in the microchannel.

  The electrode system may be a two-electrode system, a three-electrode system, or a four-electrode system, and the working electrode and the counter electrode may be disposed on the same solution side, or may be separately disposed with a molecular film therebetween.

In addition, the molecular film includes at least one kind of protein, nucleic acid, glycolipid, cholesterol, fluorescent dye, ligand, photosensitive molecule, ion channel, electron conjugated substance, co-surfactant, crown ether, fullerene, carbon Nanotubes, porphyrins, cyclodextrins, molecular tongs, polypeptides or peptides may be contained.

  The fluid 63 and the fluid 65 are preferably solutions, but may be gases. In the case of a solution, proteins, nucleic acids, nucleic acid-related substances, amyloid proteins, viruses, biological substances such as amino acids and sugars, or non-biological substances such as organic substances, inorganic substances, and ions, and these biological substances or non-biological substances The endoplasmic reticulum comprising at least one related substance may contain at least one kind.

  Moreover, the molecular film of the microreactor sensor provided with the molecular film may have a function as a diaphragm of the electrochemical cell.

  A microreactor sensor including a molecular film may have a function of controlling temperature, pressure, pH, current, voltage, magnetic field, supersonic field, light, stress, or the like in the channel.

  Further, the molecular membrane type microchannel sensor may function as a microelectrolysis cell by installing a molecular membrane containing an ion channel or a salt bridge.

  Further, in the microchannel portion where the fluid 65 stays, a molecular film may be formed on the wall surface of the microchannel and the surface of the electrode by passing the molecular film forming liquid.

  In the second embodiment, various fluids flowing to the microchannel may not be laminar.

  The fluid 67 is pure water, organic solvent, sulfuric acid, hydrochloric acid, hydrofluoric acid and nitric acid acid solution for cleaning the electrode, or an acid solution or alkaline solution containing a small amount of perchloric acid and hydrogen peroxide. Any solution may be flowed continuously in one or more types.

  In addition, the electrode pair or electrode system electrode can be renewed in the fluid 67 by chemical cleaning or electrochemical cleaning.

  The detection unit according to the second embodiment detects a change in the temperature, concentration, pH, electric signal, and change in the surface state of the molecular film in the microchannel or outside the microchannel. Can do.

  In addition, the fluid 63 or the fluid 65 containing the substance can be injected into the fluid 63 or the fluid 65 through a fine channel connected to the microchannel.

  Examples according to the second embodiment of the present invention will be described below.

(Comparative example)
As shown in FIG. 35, a solution 80a (pure water), an insulating medium 78 [mixture of dodecafluorotetradecane and tridecafluorooctanol (10: 1)], solution 80b [0.1M Tris buffer (pH = 7.5)], amphiphilic molecule mixture 79 [95% 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) + 5% Galactosylceramide (GalCer)], solution 81 [0.1 M Tris buffer (pH = 7.5)], insulation Medium 78 [mixture of dodecafluorotetradecane and tridecafluorooctanol (10: 1)] and pure water 80c, respectively, and when the solution 80b and the solution 81 reach under the electrode, the flow of the solution is stopped, The membrane potential of the molecular membrane was measured with microelectrodes placed on both sides. Further, after the membrane potential reached stable, 0.5 μl of 0.1 M Tris buffer (pH = 7.5) was injected from the sample inlet 100 as a blank sample, and the change in membrane potential was measured.

(Example)
Next, the same flow rate, volume, type of solution and solution order as described above are applied, and the solution is supplied from the sample inlet 100 to the solution 81 with 0.1 M Tris buffer solution (pH 0.5 μl) containing 25 nM HIV-1. = 7.5), and the membrane potential of the molecular film and the membrane potential change after injecting the sample containing HIV-1 were measured.

(result)
In the case of the comparative example, when the membrane potential was stabilized, no change in membrane potential was observed even when a blank sample was injected into the solution 81.

  On the other hand, in the case of the example, when a sample containing HIV-1 was injected into the solution 81, a membrane potential of several mV was observed.

(Third embodiment)
In the third embodiment, the description of the structural abnormality protein (amyloid protein), virus detection, separation, and protein refolding by the microreact provided with the molecular film described in the first embodiment. To do.

  In the third embodiment, a trace amount of a structurally abnormal protein or virus, simple, rapid, and highly sensitive detection is achieved by the interaction between an amyloid protein and a molecular membrane that is automatically, rapidly and continuously produced in a virus microreactor. A microreactor that provides a microchip and a biosensing system or is useful for early diagnosis and treatment of intractable diseases by refolding a structurally abnormal protein using a microreactor equipped with a molecular film will be described.

(Characteristics of the microreactor according to the third embodiment)
In the third embodiment, a next-generation detection chip and system for detecting a common structural abnormality protein (amyloid protein) and a virus causing various diseases and a virus with high sensitivity, a small amount, simply, and quickly are provided, and an early stage of intractable disease is provided. It is useful for diagnosis. In addition, it provides a system that can separate and remove structurally abnormal proteins from sample solutions by adsorption, deposition, and adhesion of structurally abnormal (pathogenic) proteins to molecular films, and is useful for the treatment of intractable diseases It is.

  The third embodiment is characterized in that it is useful for the treatment of intractable diseases by protein refolding due to the interaction between a structurally abnormal protein and a molecular film.

  In the third embodiment, the interaction between various proteins and drugs and lipid bilayer membranes (biological membranes), which is a kind of molecular membranes, will help to develop new drugs by elucidating the mechanisms of diseases and confirming drug effects. It is a feature.

  In other words, in the third embodiment, the structure abnormal protein (amyloid type protein) or the electrostatic film with the molecular film formed between the first fluid 26 or the second fluid 27 in the microchannel of the virus. Based on adhesion, adsorption and deposition to membranes due to interactions such as hydrophobic, hydrophilic and ionic interactions, bioaffinity, etc., and permeation change of substances contained in fluids through molecular membranes due to interactions with membranes It is a biosensor chip, biosensing system, or it is equipped with a refolding system for amyloid protein with abnormal structure, separation / removal by attaching, adsorbing, depositing and permeating amyloid protein or virus to the molecular membrane To do.

  The molecular film in the third embodiment is a monomolecular film, bimolecular film, lipid bimolecular film, reverse bimolecular film, lipid multilayer film, molecular multilayer film, micelle, reverse micelle, vesicle, reverse vesicle, emulsion. Any one of the LB films or a structure in which these are mixed is characterized.

  The amyloid type protein or virus in the third embodiment is characterized in that the first fluid 26 or the second fluid 27 contains at least one kind.

  The molecular film according to the third embodiment has at least one of a two-dimensional planar shape, a three-dimensional shape, a three-dimensional columnar shape, a conical shape, a truncated conical shape, a cylindrical shape, and an elliptical cylindrical shape. It is characterized by having.

  The molecular film according to the third embodiment is composed of at least one kind of lipid, or a lipid or protein (enzyme, molecular chaperone, antigen, antibody) modified with at least one kind of lipid and at least one kind of fluorescent molecule. , Nucleic acids, glycolipids, glycoproteins, cholesterol, fluorescent dyes, sphingolipids, ligands, polypeptides, cyclodextrins, fullerenes, carbon nanotubes, crown ethers, steroids, porphyrins, ion channels, electron conjugated substances, redox agents, etc. This is a molecular film comprising the above molecules, and is characterized in that a microdomain is formed in the molecular film.

  The microdomain in the third embodiment is partially formed in the formed molecular membrane, particularly in the lipid bilayer in the microchannel. Alternatively, a microdomain-like functional film is formed in the microchannel.

  The molecular film according to the third embodiment includes a first fluid 26 and a second fluid through a hollow polygonal inlet such as a link, a hollow ellipse, a hollow corner, a hollow hexagon, or the like in the channel. 27 or between the fluid and the channel wall.

  The inlet of the amphiphilic molecule mixture, the first fluid 26 and the second fluid 27 in the third embodiment has a three-dimensional structure of a ring shape, an ellipse shape, a semicircular shape, a square shape, or an unequal polygonal shape. It is characterized by having.

  The virus in the third embodiment is an icosahedral virus consisting only of a capsid and a nucleic acid, a virus having a coating called an emperope outside the capsid, and an envelope outside the capsid in which the capsomer is spirally connected. It is characterized by having a virus and hepatitis B virus.

  In addition, among the components extracted from the virus, in the case of anions, contaminating proteins such as capsids are extracted and removed by reverse micelles composed of a cationic surfactant. Alternatively, a negatively charged contaminating protein is adsorbed and separated by a lipid bilayer membrane having a positive charge.

  By these methods, contaminating proteins extracted from viruses and DNA can be separated, and DNA after removing the contaminating proteins can be detected by a DNA sensor without being affected by the contaminating proteins. .

(Structure of the analysis system according to the third form)
The analysis system according to the third embodiment of the present invention is the same as the microreactor analysis system (1) to the microreactor analysis system (4) according to the first embodiment shown in FIGS. The description is omitted here.

  FIG. 36 is a schematic diagram of a microreactor constituting the production / regeneration unit 11 and the reaction unit 14 shown in FIG. As shown in FIG. 36, the microreactor 1 has an outlet including a third inlet 21, a first inlet 22, and a filter 41. The filter 41 has a characteristic of transmitting an aqueous solution and a low molecular weight substance, but not transmitting a high molecular weight protein, polypeptide, peptide or virus. Further, depending on the type of the filter 41, it is very effective for concentration of amyloid protein and virus present in a minute amount in a specific sample and sample solution, or for highly sensitive detection. The microreactor 1 can be used repeatedly as a microsensor chip and is very economical.

(sample)
Samples of the analysis system according to the third embodiment are structurally abnormal proteins and viruses. In addition, proteins, polypeptides, peptides, membrane-bound drugs, antibiotics, and the like that cause the formation of pores in the membrane by interaction with the molecular membrane can also be used as samples. Examples of these samples include alamethicin, gramicidin, and bee venom melittin.

  In addition, a substance that can cause changes in the electrochemical characteristics of the film or the surface structure and fluorescence of the film by the interaction with the molecular film can be used as the sample. Examples of such samples include toxic substances such as cholera toxin, tetanus (tetanus toxin), botulinum toxin, dioxin, arsenic (arsenic), tetrodotoxin, ciguatoxin, α-bungarotoxin, ricin and saxitoxin. Antibiotics include penicillin G, kanamycin A, tetracycline, spretomycin and the like.

(Disease caused by protein structure abnormality and viral infection)
Diseases caused by structurally abnormal proteins are called conformation diseases or folding diseases. One of conformational diseases is amyloid disease (or amyloid-cis), which is formed from amyloid-type protein or a peptide having about 10 residues, and is caused by the deposition of amyloid fibers.

  The function of amyloid-type protein is closely related to its higher-order structure, and the structural change of the protein due to external environmental conditions impairs the original biological function, and amyloid fiber formation and amyloid fiber deposition cause mad cow disease, human Prion diseases such as Creutzfeldt-Jakob disease, Kourou disease, sheep scrapie, Alzheimer's dementia disease (Aβ peptide, amyloid β protein), Dialysis amyloid-cis (β microglobulin), Medullary thyroid cancer (calcitonin), AL amyloidosis It is known to cause (immunoglobulin light chain), familial amyloid polyneuropathy (transthyretin), familial amyloid-cis (lysozyme), Parkinson's disease (α-synuclein), Huntington's disease (huntingtin) and the like. The parentheses indicate the causative protein.

  The above-mentioned amyloid-type proteins are molecular membranes, particularly lipid bilayer membranes, microdomains (rafts and gaveolaes) formed in lipid bilayer membranes, anchor proteins contained in or projected on lipid bilayer membranes (GPI anchor proteins) ), Receptor proteins, glycoproteins, sphingolipids (gangliosides), glycolipids, antigens, specific interactions with antibodies, or electrostatic, hydrophobic, hydrophilic and ionic interactions with membranes If it is an amyloid protein or its precursor protein that causes a permeation change of the substance through the membrane due to adsorption, adhesion, deposition, and formation of a pore that penetrates the membrane due to the interaction with the membrane, it is particularly limited to the above-mentioned types There is nothing.

  The above-mentioned microdomain is a microdomain formed in a lipid bilayer, and is a microdomain formed using a membrane region formed by sphingomyelin, cholesterol and sphingolipid (ganglioside) as a base (13 to 70 nm). It is. It is also considered that these lipids aggregate in a phospholipid membrane with many unsaturated fatty acid chains to form an ordered liquid crystal microdomain (raft). In addition, MAL, flottillin, and ESA, which are intramembrane proteins, are proteins constituting a microdomain (raft).

(Amyloid protein detection principle and means)
The principle of a biosensor based on the interaction between these proteins and molecular membranes, particularly lipid bilayer membranes, will be described below by taking amyloid proteins that cause prion disease and Alzheimer's dementia as examples.

  First, the prion disease and its detection principle and means are described. As a common pathogen such as mad cow disease, human Creutzfeldt-Jakob disease, Kuru disease, sheep scrapie, prion which is a kind of protein can be considered. Therefore, these diseases are collectively called prion diseases. The prion has two structures, an α helix and a β sheet. The prion of the α helix is a normal prion, but the β sheet prion is a protein structure abnormal prion. When a normal α-helix prion is infected with an abnormal β-sheet prion, the structure is successively changed to a β-sheet using β-sheet prion as a template.

  In vivo, α-prion and β-prion eventually undergo proteolysis, but α-prion is completely degraded, but β-prion is not completely degraded and N-terminal 67 residues By losing it, it aggregates further and forms amyloid plaques. This amyloid plaque is considered to be a direct cause of neurodegeneration specific to mad cow disease.

  On the other hand, different prion structures have different hydrophilic or hydrophobic properties. Normal α-helix prions are hydrophilic, while abnormal β-sheet prions are hydrophobic. Therefore, it is clear that the interaction of these molecular membranes, particularly lipid bimolecular membranes and bimolecular lipid membranes forming rafts, varies depending on the hydrophilic or hydrophobic properties of prions resulting from the difference in structure.

  In the third embodiment, the membrane potential, impedance, dielectric caused by the difference in the interaction between the molecular membrane of prion caused by the difference in structure, particularly the lipid bilayer membrane or the microdomain formed in the lipid bilayer membrane. It is to discriminate between normal α-prion and abnormal β-prion by detecting and observing changes in the rate, changes in material permeation through the membrane, or changes in the surface structure and state of the membrane.

  Specifically, for example, normal α-prion interacts with a POPG membrane (an abbreviation of palmitoyloleoyphosphatidylglycerol), but the membrane is not destroyed. On the other hand, abnormal β-prion similarly interacts with the POPG film, but the film is destroyed. Normal α-prion does not interact with raft membranes composed of DPPC (dipalmitoylphosphatidylcholine), cholesterol, and sphingomyelin, but abnormal β-prion interacts with raft membranes, but the membrane is stable. Can be maintained.

  That is, in the third embodiment, the difference in membrane potential caused by the interaction with the molecular membrane of prion, particularly the lipid bilayer membrane or the microdomain formed in the lipid bilayer membrane, It is characterized in that abnormal prions are recognized by detecting changes in the permeation substance or observing the surface state of the membrane.

  Next, Alzheimer's dementia and its detection principle and means are described. Alzheimer's dementia is also considered to be due to the formation of amyloid (aggregation) fibers that significantly increase aggregation due to abnormal protein structure, as in prion disease. This amyloid fiber aggregates abnormally in cells of the central nervous system, resulting in cell loss of the central nervous system, leading to dementia.

  On the other hand, the causative proteins of Alzheimer's dementia are Aβ peptide and amyloid β protein. In particular, amyloid β protein has strong aggregation in an aqueous solution, and when incubated at a concentration of several tens of μM or more, amyloid fibers having a β sheet structure are easily obtained. Formed. On the other hand, the physiological amyloid β protein concentration in the living body exists at a very low concentration on the order of nM and is soluble, so that spontaneous aggregation of the protein does not occur, so that early diagnosis and treatment are difficult.

  In the third embodiment, a small amount of amyloid β protein is produced by a specific interaction between amyloid β protein and a microdomain consisting of sphingomyelin, cholesterol and sphingolipid (ganglioside) present in the lipid bilayer, particularly ganglioside. By utilizing the characteristic that a small amount of amyloid β protein is aggregated in this species, a seed of the amyloid β protein is formed in the micro domain by attachment and deposition to the micro domain.

  That is, Alzheimer detects and observes large changes in electrical and surface characteristics of the membrane or permeation of substances through the membrane due to aggregation of amyloid β protein into microdomains existing in the lipid bilayer membrane. Perform early diagnosis of dementia.

(Type of virus)
As a virus that is a sample according to the third embodiment of the present invention, an icosahedral virus consisting only of a capsid and a nucleic acid, a virus having a film called an envelope on the outside of the capsid, and a capsomer formed in a spiral shape There is a virus with an envelope outside the capsid. Examples of the icosahedron virus include poliovirus that causes motor paralysis and hepatitis A virus that causes hepatitis. Examples of viruses having a film called an envelope on the outside of the capsid include human immunodeficiency virus (HIV), Sindobivirus, human herpes virus type 6 and human herpes virus type 8. In addition, as a virus having an envelope outside the capsid in which the capsomer is spirally formed, there is a virus such as SARS virus. In addition, there are three types of viruses such as hepatitis B virus.

  As described above, the types and forms of viruses vary, but molecular membranes, particularly lipid bilayer membranes, microdomains (rafts and gaveolaes) formed in lipid bilayer membranes, are projected on the surface of lipid bilayer membranes. Anchor protein (GPI anchor protein), receptor protein, glycoprotein, sphingolipid (ganglioside), glycoprotein, glycolipid, antigen, specific to antibody, electrostatic to membrane, hydrophobic, hydrophilic and ionic Is a virus that causes permeation change of the solution phase material to the membrane by the adsorption, adhesion, deposition on the membrane and the formation of pores that penetrate the membrane due to the interaction. If so, it is not particularly limited to the type of virus.

(Virus detection principle and means)
In the third embodiment, viral molecular membranes, in particular lipid bilayer membranes, microdomain-like membranes, microdomains formed in lipid bilayer membranes (raft, gaveola), and protrusions on the surface of lipid bilayer membranes Anchor protein (GPI anchor protein), receptor protein, glycoprotein, sphingolipid (ganglioside), glycoprotein, glycolipid, antigen, specific with antibody, electrostatic with membrane, hydrophobic, hydrophilic and ionic, etc. Due to the interaction of the membrane with the changes in membrane potential, impedance, dielectric constant, absorption spectrum, fluorescence spectrum and membrane surface state caused by adsorption, adhesion, deposition, fusion and interaction with the membrane, or the above interaction Utilizing the feature that causes the permeation change of the substance to the membrane by the formation of pores through the accompanying membrane To.

  In the third embodiment, changes in membrane potential, current, impedance, dielectric constant, electric capacity, viscosity, density, refractive index, absorption spectrum, fluorescence spectrum, etc. associated with the interaction described above, It is detecting a virus by detecting a change or observing a change in the surface structure and state of a film.

(Measurement device and detection method)
Various electrical, chemical and physical changes caused by the interaction of the sample with the molecular film can be specifically detected by the following detection apparatus and method.

  For example, optical microscope, electron microscope, laser electron microscope, differential interference phase contrast microscope, scanning electron microscope (SEM), total reflection Fourier transform infrared analyzer, laser Raman spectrometer A fluorescence microscope, a polarizing microscope, an X-ray analyzer, an atomic force microscope (AFM), and the like can be used, but the apparatus used in the present invention is not particularly limited to these apparatuses.

  As the characteristics of the molecular film according to the third embodiment, for example, the physical properties such as the fluidity and packing state of the film, the charge density of the film surface, the hydrophobic and hydrophilic characteristics are the film potential, the impedance of the film, and the potential. It can be examined by measuring the dielectric constant of the capacitance, film or solution.

  Devices that measure electrical signals such as membrane potential and current include, for example, electrometers, digital multimeters, potentiostats, etc., but high-sensitivity electrometers with high resistance, low-pass filters, amplifiers that can amplify signal electrical signals, etc. Is desirable. As an apparatus for measuring the impedance and capacitance of the membrane, an impedance analyzer, an LCR meter, a vector impedance meter, and the like can be used, but a potentiostat, in particular, a signal electrical signal smaller than a picoampere can be measured and a high-speed sweep can be performed. It is desirable to use in combination with a device such as an ultra-sensitive potentiostat equipped with a low-pass filter. Bipotentiostats (dual potentiostats) can be used as other electrochemical measurement devices, but the devices used in the present invention are not particularly limited to these devices.

  In order to prevent electrical noise, it is desirable to perform the above measurement in a shield box.

(Measuring method of changes in properties of membrane and fluid due to interaction of matter with membrane)
On the other hand, biologically related substances such as polypeptides, saccharides, proteins, and viruses are attached to the membrane due to the interaction with the membrane, adsorbed, deposited, and the change in fusion depends on the device and method for detecting the characteristics of the membrane and the surface condition of the membrane An observation device can be used.

  Furthermore, membrane permeation changes of substances such as ions, redox species, organic substances, saccharides and amino acids contained in the fluid due to the interaction with the molecular film of amyloid type protein or virus, polypeptides, saccharides, bitter substances, sweet substances, Indirect or direct changes in permeation of substances such as proteins and substances such as molecules into membranes, electrochemical changes in membranes and fluids, absorption spectra, fluorescence spectra, or changes in membrane surface properties Can be detected and observed.

(Measuring method of substance membrane and fluid by permeation of substance membrane and interaction with membrane)
Furthermore, as a method for directly detecting ions, redox species, molecules and substances that have permeated through the membrane, the following methods can be taken. For example, an electrode such as an ion sensor, a microelectrode, or a sensor electrode that is a sensor terminal of an electrometer, a potentiostat, and a galvanostat device is installed inside or outside the microchannel, and the local, in-situ, Detection can be performed. Furthermore, these membrane-permeable substances can be collected and measured at the outlet of the channel.

  On the other hand, depending on the nature of each substance, the membrane permeating substances flowing out from the channel are pH meter, viscometer, density meter, refractometer, absorptiometer, high performance liquid chromatography (HPLC), liquid chromatography (LC ), Gas chromatography (GC), gas chromatography mass spectrometer (GC / MS), thin layer chromatography analyzer (TLC), fluorescence spectrometer, Fourier transform infrared spectrometer (FTIR), and other chemical analyzers Can be used to analyze the corresponding component.

  For example, as described above, if the material that permeates the membrane does not cause a change in the fluorescence spectrum, it is qualitatively and quantified by measuring the electrical signal, pH, viscosity, density, refractive index, absorption spectrum, and thermal signal of the permeable material. be able to.

  On the other hand, when the substance that has passed through the membrane causes fluorescence spectrum conversion in the membrane and fluid, the substance can be qualitatively and quantitatively detected by detecting changes in the fluorescence spectrum.

(Peptide, polypeptide and protein analyzer)
For analysis of peptides, polypeptides and proteins, high performance liquid chromatography (HPLC), spectrophotometry and circular dichroism spectroscopy can be used.

  The secondary structure of the protein can be analyzed using a Fourier transform infrared spectrometer.

(Operation and effect of the microreactor according to the third embodiment)
According to the microreactor according to the third embodiment, various electrical and chemical effects caused by the interaction between a structurally abnormal protein (amyloid protein) and a stationary or flowing molecular film produced in a microchannel of a virus. It is possible to detect structural and physical changes, and to detect a structurally abnormal protein (amyloid protein) and a virus with high sensitivity and speed. Furthermore, rapid refolding of structurally abnormal proteins can also be performed.

  The microreactor and the analysis system according to the third embodiment form a molecular film having fluidity between liquid / liquid laminar flow, gas / liquid laminar flow, or solid / laminar flow. It is characterized in that it is used as a sensor for various samples by utilizing the biological membrane function possessed by. Furthermore, it is possible to construct a sensor having various functions with a single chip by utilizing the fluidity of the membrane, and at the same time, the sensor can be repeatedly used.

  Next, an example of the analysis system according to the third embodiment of the present invention will be described.

Example 1
In Example 1, amyloid type protein (Aβ) was detected. The lipids, solutions, and proteins used in Example 1 are as follows.

Lipid: Egg yolk phosphatidylcholine lipid solution containing GM1 / sphingomyelin / cholesterol (40:30:30): 0.1 M Tris buffer (pH 7.5)
Protein: 0.6μM Amyloid β protein (Aβ)
As shown in FIG. 37, a solution of egg yolk phosphatidylcholine lipid containing GM1 / sphingomyelin / cholesterol (40:30:30) is introduced at a predetermined flow rate from the third inlet 21 of the microreactor which is kept at 25 ° C. Then, 0.1 M Tris buffer (pH 7.5) is introduced from the first inlet 22 and the second inlet 23 at a predetermined flow rate, respectively, and further, 0.6 μM amyloid is intermittently introduced from the second inlet 23. A 0.1 M Tris buffer (pH 7.5) containing β protein (Aβ) was introduced.

(result)
When 0.1 M Tris buffer (pH 7.5) containing amyloid β protein (Aβ) was intermittently flowed, changes in membrane potential were detected by electrodes placed on both sides of the membrane.

  The experimental results of Example 1 showed that the microreactor equipped with a molecular film can specifically detect amyloid β protein (Aβ).

(Example 2)
In Example 2, this was performed as a model system for refolding denatured protein using a microreactor equipped with a molecular film. The refolding was performed using carbonic anhydrase as a denatured protein model. The surfactant, organic solvent, and protein used in Example 2 are as follows.

Surfactant: 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)
Organic solvent: decane denatured protein: carbonic acid dehydrogenase As shown in FIG. 38, 3 μM denatured carbonic acid dehydrogenation at a predetermined flow rate from the first inlet 22 and the second inlet 23 of the microreactor kept at 25 ° C. A 0.1 M Tris buffer (pH 7.5) containing an enzyme and 0.1 M guanidine hydrochloride was introduced, and 7% 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine was introduced at a predetermined flow rate from the third inlet 21. A decane solution containing was introduced to perform the refolding operation.

(result)
The denatured carbonic acid dehydrogenase was rapidly refolded by contact with the molecular film formed in the microreactor.

(Example 3)
In Example 3, an amphiphilic molecule mixture of palmitoyl oleoyl phosphatidylcholine (POPC) was introduced at a predetermined flow rate from the third inlet 21 of the microreactor having a constant cell structure as shown in FIG. A 0.1 M Tris buffer (pH 7.5) was introduced at a predetermined flow rate from the first inlet 22 and the second inlet 23, respectively. Further, 0.1 M Tris buffer (pH 7.5) containing 0.6 μM β-type prion protein was intermittently introduced from the second inlet 23.

(result)
When 0.1 M Tris buffer (pH 7.5) containing 0.6 μM β-type prion protein was flowed, changes in membrane potential were detected by electrodes placed on both sides of the membrane.

(Comparative Example 3)
On the other hand, when a Tris buffer solution (pH 7.5) containing 0.6 μM α-type prion protein was intermittently passed from the second inlet 23 under the same experimental conditions as described above, no change in membrane potential was detected. .

  From the experimental results of Example 3 and Comparative Example 3, it was shown that the microreactor equipped with a molecular film can specifically detect β-prion protein.

Example 4
In Example 4, extraction of viral RNA and reverse transcriptase in HIV-1 with a serum-containing solution, membrane potential change, and detection of HIV-1 using a gene sensor and protein sensor were performed.

  As shown in FIG. 39 (a), an amphiphilic molecule mixed solution containing sphingomyelin, galactosylceramide (GalCer) and cholesterol is flowed from the third inlet 21 of the microreactor kept at 25 ° C. at a predetermined flow rate. A 0.1 M Tris buffer (pH 7.5) was introduced from each of the first inlet 22 and the second inlet 23. Further, a solution containing serum and HIV-1 was intermittently introduced from the second inlet 23.

  A microdomain (raft) -like film is formed between the liquid / liquid laminar flow in the microchannel by the amphipathic molecule mixed solution (lipid mixture 25) flowed from the third inlet 21, and this microdomain-like film is formed. The membrane interacts specifically with the site of HIV-1 Gp120 glycoprotein V3 loop {see Fig. 39 (b)}, resulting in membrane fusion between HIV and lipid bilayer, and within HIV-1. The HIV-1 viral RNA and reverse transcriptase {see Fig. 39 (c)} were released on the opposite side of the membrane {see Fig. 39 (a)}. On the other hand, since serum does not permeate the membrane, it flows out from the outlet as it is and can be separated from HIV-1 viral RNA and reverse transcriptase.

(result)
As mentioned above, viral RNA, reverse transcriptase and serum can be separated by membrane fusion between HIV-1 and lipid bilayer membrane, and at the same time, electrical signals such as changes in membrane potential (potential, resistance, etc.) can be obtained by membrane fusion. , Current, dielectric constant, etc.) could be detected by electrodes installed in each laminar flow.

  Further, the extracted HIV-1 transferase and RNA could be detected by a protein sensor or gene sensor without being affected by serum.

(Fourth embodiment)
In the fourth embodiment, a microreactor for producing a reverse micelle molecular film will be used to explain normal extraction and reverse extraction of a substance, particularly a biological substance, or chemical reaction accompanying extraction and refolding of a denatured protein.

  In the fourth embodiment, high-efficiency and rapid extraction of substances, high-activity, high-efficiency and rapid extraction of bio-related substances such as proteins, simple, short-time, energy-saving and mild reaction / extraction conditions; It is an object of the present invention to provide a micro extraction system (reverse micelle type microreactor) using reverse micelles that performs rapid refolding.

(Features of the microreactor according to the fourth embodiment)
The fourth embodiment is characterized in that the problem in conventional reverse micelle extraction is greatly improved by performing reverse micelle extraction in a microreactor.

  Specifically, the present invention can increase the oil-water interfacial area without agitation as in the case of conventional reverse micelle extraction, so that rapid extraction of useful substances can be achieved without the emulsification of the surfactant. It is characterized by doing.

  In the fourth embodiment, it is easy to control the reverse micelle size by the micro space, and the extraction by the stable micelle structure by the laminar flow contact with oil / water is possible. Therefore, compared with the conventional reverse micelle extraction, It is characterized by performing extraction with high selectivity and rapid extraction.

  In the fourth embodiment, the extraction operation can be performed under mild conditions. Therefore, the extraction of relatively small biomolecules such as proteins / enzymes (occupying about 20% of cells), amino acids, peptides, nucleic acids, etc. And a biologically active substance that reaches biological particles such as microbial cells, and a substance separation / extraction system.

  The fourth embodiment is characterized in that the three-dimensional structure and activity of a denatured protein are recovered using a micro extraction system (reverse micelle type microreactor) using reverse micelles.

  The fourth embodiment is characterized by providing a bioreactor system using a micro extraction system (reverse micelle type microreactor) using reverse micelles.

  In the fourth embodiment, a micro extraction system using reverse micelles is configured such that substances in the raw water phase are transferred to the reverse micelles existing in the reverse micelle solution phase between the laminar flow of the raw water phase and reverse micelle solution phase in the microchannel. Step 1 in which protein is refolded in the normal extraction or in association with extraction, step 2 in the recovery or recycling of the aqueous raw material solution, and back extraction of the substance and the refolded protein from the reverse micelle solution extracted in the normal manner It comprises a step 3, a circulation step 4 for reusing the reverse micelle solution, and a step 5 for recovering the extracted product.

  That is, the fourth embodiment contains one or more types of amphipathic molecules and ligands, phospholipids, glycolipids, cholesterol, proteins, sphingolipids, redox species, cell recognition molecules, receptors, and a small amount of water. A microchannel for flowing a reverse micelle solution composed of one or more types of amphipathic molecules, and an aqueous raw material solution containing at least one kind from substances such as biological materials, various molecules, redox agents, molecular chaperones and metal ions The two liquid phases flowing out from both channels are transported in a certain direction while maintaining a laminar flow state in the same microchannel. At this time, both liquid phases of the reverse micelle solution phase and the raw water phase are mixed. Normal extraction of biological materials, etc. into reverse micelles by mutual contact at the interface, chemical reaction accompanying extraction, protein refolding Step 1 is performed, followed by a raw material aqueous solution recovery or recirculation channel, a raw material aqueous solution recovery or recirculation step 2, and further, bio-related substances, various molecules, ions, etc. A reverse micelle solution channel incorporating a plurality of recovered aqueous phase channels for reverse extraction, a step 3 for reverse extraction, and a circulation step 4 for reusing the reverse micelle solution; And Step 5 for recovering the extracted product.

  Step 3 further includes step 3 (a), in which the biologically relevant substance is back-extracted into the recovered aqueous phase by contact between the laminar flows of the reverse micelle solution phase and the recovered aqueous phase that are extracted in the forward direction. Step 3 of performing reverse extraction by controlling the temperature of the biological substance in the recovered aqueous phase by controlling the temperature of both liquid phases in the channel in the reverse micelle solution phase and the recovered aqueous phase contained with a micro control chip. (B), and a step 3 (c) for back-extracting the biological substance to the recovered aqueous phase by introducing a hydrophilic organic solvent from the branch channel into the channel of the reverse micelle solution. is there.

  The reverse micelle of the fourth embodiment is a reverse micelle composed of at least one kind of amphiphilic molecule, at least one kind of amphiphilic molecule, at least one kind of co-surfactant, ligand, phospholipid, It is a mixed reverse micelle composed of glycolipid, cholesterol, protein, sphingolipid, redox species, cell recognition molecule, and receptor.

  A water pool containing a water pool or a redox species, a nucleic acid-related substance, and a protein is present at the center of the reverse micelle according to the fourth embodiment, and a biological substance is taken into the water pool.

  The recovered aqueous phase in step 3 in the fourth embodiment has a different pH and salt concentration.

  The aqueous raw material solution according to the fourth embodiment is characterized by containing substances, in particular, proteins, denatured proteins, amino acids, polypeptides, peptides, nucleic acids, nucleic acid-related substances, plasmids, bacteria, plant cells, and the like that are biologically related substances. To do.

  In the fourth embodiment, a redox agent, a molecular chaperone, and a metal ion are incorporated into a reverse micelle water pool containing a denatured protein, and the three-dimensional structure and activity of the protein are restored in the reverse micelle water pool. It is characterized by.

(Structure of micro extraction system using reverse micelle)
As shown in FIG. 40, the microreactor system (reverse micelle type microreactor) according to the fourth embodiment is configured to perform normal extraction and recirculation of substances such as biological substances using a microextraction system (reverse micelle type microreactor) using reverse micelles. A normal extraction unit 91 for performing folding, a raw material recovery unit 92 for recovering an aqueous raw material solution, a reverse extraction unit 93 for performing reverse extraction of substances such as biological substances extracted by normal extraction, and reverse micelles A circulation unit 95 for circulating the solution and a product recovery unit 94 for recovering each extraction product obtained by back extraction are provided. These constituent parts can be freely combined corresponding to the object of normal extraction and reverse extraction.

  For example, FIGS. 41 and 42 show schematic diagrams of protein refolding performed after positive extraction and positive extraction of different biologically relevant substances (or substances) in the positive extraction unit 91, for example.

  As shown in FIG. 41, the positive extraction microchannel has a sixth inlet 96 for introducing a solution containing reverse micelles (RM) for extracting different biological substances (or substances) into the reverse micelles and the biological relations. There is a seventh inlet 97 for introducing a raw material aqueous solution (W) containing substances (P1, P2, P3) and the like, and these two channels are connected to the microchannel (3). In the microchannel 3, both laminar flow phases are formed, and biologically relevant substances (P1, P2, P3) contained in the raw material aqueous phase are positively extracted into reverse micelles (RM) through the interface between both laminar flows.

  In this case, the reverse micelle is a reverse micelle composed of one or more types of amphiphilic molecules, or at least one type of amphiphilic molecules and at least one type of ligand, phospholipid, glycolipid, cholesterol, protein, sphingolipid, oxidation A mixed reverse micelle consisting of a reducing species, a molecular chaperone, a cell recognition molecule, a co-surfactant and a receptor. In addition, a small amount of water, redox species, metal ions, various molecules, and biological substances can be contained in the reverse micelle in advance. Various substances constituting the reverse micelle and various substances included in the reverse micelle will be described in detail later.

  On the other hand, when the raw material aqueous phase (W) contains a denatured protein, the denatured protein can be refolded as shown in FIG. First, denatured protein is extracted into reverse micelles (RM). Next, reverse micelle (RM-Ox / Red) containing an oxidizing agent and a reducing agent, a molecular chaperone, adenosine triphosphate, and a metal ion are included from a branch channel (10th inlet 104) of the reverse micelle solution phase. Reverse micelles (RM-Cpn / ATP / M) can be added to refold denatured proteins.

  The raw material recovery unit 92 recovers an unreacted raw material aqueous solution.

  The back extraction unit 93 performs back extraction of the biological material extracted by the normal extraction unit and the protein after refolding. As shown in FIG. 43, the back extraction unit 93 performs the first back extraction by controlling the conditions such as the salt concentration and pH of the aqueous solution (recovered aqueous phase) or adding a ligand reagent in the aqueous solution (recovered aqueous phase). Back extractor 93a (step 3a), third back extractor 93b (step 3b) that performs back extraction by controlling the temperature in the microchannel, hydrophilic organic substances such as alcohols in the reversely extracted reverse micelle solution phase A third back extraction unit 93c (step 3c) that performs back extraction into the recovered aqueous phase by adding a solvent is included.

  The product recovery unit 94 recovers the extraction product obtained by the back extraction unit 93.

  The circulation unit 95 circulates the reverse micelle solution from the back extraction unit 93 to the normal extraction unit 91.

  The shape of the microchannels used in the forward extraction unit 91 and the reverse extraction unit 93 according to the fourth embodiment is preferably used by making the straight microchannels into a multi-tube type in order to reduce frictional resistance. However, curved microchannels can be used as appropriate according to the application. The microchannel diameter and shape of the microchannel are not particularly limited as long as the microchannel according to the fourth embodiment can form a laminar flow of the reverse micelle solution phase and the aqueous phase.

  Note that the material of the microchannel is the same as that in the first embodiment, and thus the description thereof is omitted here.

(Substances used in micro extraction systems using reverse micelles)
Since the substances such as amino acids, peptides, and nucleic acid-related substances used in the micro extraction system (reverse micelle type microreactor) using the reverse micelle according to the fourth embodiment are the same as those in the first embodiment, here Description is omitted. In addition, proteins, denaturing agents, oxidizing agents and reducing agents, surfactants, and organic solvents, for example, are used as follows, but are not particularly limited thereto.

  Proteins include acyl-CoA oxidase, acyl-CoA synthetase, ascorbate oxidase, asparaginate aminotransferase, aspartate β-decarboxylase, aspartase, acetate kinase, aminoacylase, amino acid oxidase, aminopeptidase, amylase, alanine dehydration Elementary enzyme, arabanase, arabinosidase, RNA polymerase, alkaline xylanase, alkaline cellulase, alkaline protease, alkaline lipase, alcohol dehydrogenase, aldehyde dehydrogenase, aldolase, α-acetolactic decarboxylase, α-chymotrypsin, isoamylase, isocitrate dehydrogenase , Invertase, uricase, urease, urokinase, esterase, N-acetyl Neuraminate lyase, endo-β-glucanase, ω-hydroxylase, catalase, carboxylesterase, carboxypeptidase, carbonic anhydrase, γ-glutamine transpeptidase, xanthine oxidase, formate dehydrogenase, xylanase, xylan acetylesterase, xylose isomerase, chymosin , Guanosine 5'-phosphate synthetase, citrate synthetase, glycerol oxidase, glycerol kinase, glycerol 3-phosphate oxidase, glucoamylase, glucosidase, glucosyltransferase, glucose isomerase, glucose-1-oxidase, glucose oxidase, glucose dehydrogenase Glucose dehydrogenase, glucose 6-phosphorus Dehydrogenase, glutamate decarboxylase, glutamate dehydrogenase, creatininase, creatinine deiminase, creatinase, chloroperoxidase, 5'-adenylate deaminase, colipase, cholinesterase, choline oxidase, cholesterol oxidase, thermolysin, sarcosine oxidase, sarcosine dehydrogenase, 3α-hydroxysteroid dehydrogenase, 3-chloro-D-alanine chloride, diaphorase, cyanate aldolase, cyclodextrin glucosyltransferase, dihydropyrimidinase, streptokinase, superoxide dismutase, subtilisin, cephalosporin acylase, cephalosporin amidase , Cellulase, cellobi Ohydrolase, cytochrome C, thymidylate synthase, DNA polymerase, deoxyribose-5-phosphate aldolase, dextranase, dopa decarboxylase, transglutaminase, triose phosphate isomerase, trypsin, tryptophanase, tryptophan synthetase, naringinase, nitrile Hydratase, lactate dehydrogenase, neuraminidase, halohydrin epoxidase, halohydrin halogen halide lyase, haloperoxidase, histidine ammonia lyase, hydantoinase, pyranose-2-oxidase, pyruvate oxidase, phenylalanine ammonia linase, phenol oxidase, Tressin oxidase, flavin enzyme, purine nucleoside phosphorylase Pullulanase, protease, prourokinase, proteinase, proline iminopeptidase, bromoperoxidase, hexokinase, pectinase, pectinesterase, pectin transeliminase, β-etherase, β-galactosidase, β-glucanase, β-glucoamylase, β-fructofurano Sidase, β-fructofuracinase, peptidase, hemicellulase, penicillin amylase, penicillin amidase, peroxidase, pentosanase, phosphodiesterase, phospholipase, phosphorylase, polygalacturonase, mannanase, mutanase, mutarotase, lactase, lactonohydrolase, lactoperoxidase, Rakumaze, racemase, laccase, lyase, ligase, Gunin peroxidase, lysyl endopeptidase, lysine oxidase, lysine decarboxylase, lysozyme, lipase, ribulose 1, 5-bisphosphate carboxylase, lipoprotein lipase, ribonuclease A, malate dehydrokinase, luciferase, leucine aminopeptidase, rhodanase, albumin, Insulin, interferon, interleukin, γ-globulin, chymotrypsinogen, concanavalin A, bacteriorhodopsin, proinsulin, β-lactoglobulin, hemoglobin, myoglobin, trypsin inhibitor, rhodopsin and the like can be used.

  The denaturing agent is not particularly limited as long as it is a denaturing agent that can solubilize the aggregated protein in an aqueous solution. For example, denaturing agents such as guanidine hydrochloride, urea, dithiothreitol (DTT), 2-mercaptoethanol, and cysteine can be used.

  The oxidizing agent and reducing agent are not particularly limited as long as they can refold denatured protein extracted into reverse micelles. For example, oxidized glutathione (GSSG), cystine and As the reducing agent, such as dithiothreitol (DTT), reduced glutathione (GSH), dithiothreitol (DTT), cysteine, 2-mercaptoethanol, and the like can be used.

The molecular chaperone is not particularly limited as long as it can refold a denatured protein extracted into reverse micelles. For example, chaperones (GroEL, Hsp60), Hsp70 (Dnak), Hsp90 (HtpG), HSp104 ( CIPB) etc. (in parentheses are the names for E. coli)
In addition, as surfactants, there are cationic surfactants, anionic surfactants, amphoteric surfactants, nonionic surfactants, etc., but cells with molecular weights around 100 to several microns There is no particular limitation as long as the above substances can be solubilized. Specific examples include alkyl quaternary ammonium salts (CTAB, TOMAC, etc.), alkyl pyridinium salts (CPC, etc.), dialkyl sulfosuccinates (AOT, etc.), dialkyl phosphates, alkyl sulfates (SDS, etc.), alkyl sulfonic acids. Examples include salts, polyoxyethylene surfactants (Tween, Brij, Triton, etc.), alkyl sorbitans (Span, etc.), lecithin surfactants, betaine surfactants, etc. It is not something. In addition, an auxiliary surfactant can be used to stabilize the structure of the reverse micelle.

  Solubilization with ionic surfactants is often accompanied by denaturation of membrane proteins, but when non-ionic surfactants are used, they are solubilized without losing the biological activity of membrane proteins. The use of nonionic surfactants is desirable.

  In reverse micelle extraction, polyols (such as glycerol), reducing agents (such as dithiothreitol), chelating agents (such as EDTA), and protease inhibitors (such as trypsin ion inhibitors) are used to stabilize membrane proteins. May need to be added.

  The solvent constituting the reverse micelle solution may be an organic solvent. When using an organic solvent, for example, aliphatic hydrocarbons such as hexane, heptane, octane, isooctane and decane, aromatic hydrocarbons such as benzene and toluene, cycloaliphatic hydrocarbons such as cyclohexane, halogenation such as chloroform and dichloroethane Aliphatic hydrocarbons and the like can be used, but are not particularly limited thereto.

  Moreover, a reverse micelle solution can be prepared using, for example, a supercritical fluid or a high-pressure fluid instead of an organic solvent. For example, a supercritical fluid such as ethane, propane, carbon dioxide, or a high-pressure fluid can be used, but it is not particularly limited thereto.

(Operation and effect of micro extraction system using reverse micelle)
According to the micro extraction system (reverse micelle type microreactor) 2 using reverse micelles according to the fourth embodiment, the following operations and effects are obtained.

1) Since rapid and highly efficient extraction is possible, protein denaturation / inactivation during the extraction operation can be suppressed.

2) Since it does not require power for stirring, it is energy saving.

3) Since highly efficient extraction can be realized, it is an environmental load reduction type with little waste.

4) Since the microchannel diameter is small, temperature control of the entire system is easy.

5) Extraction can be performed with a stable reverse micelle structure by laminar flow contact.

6) Since the oil / water interface can be enlarged without stirring, useful substances can be rapidly and continuously extracted without the emulsification of the surfactant.

7) Refolding and extraction of denatured protein can be performed with one chip.

8) Utilizing selection and extraction characteristics of various substances and optical isomers of reverse micelles, and further combining with various analytical instruments, it can be used as a new sensor.

  Next, an example of the analysis system according to the fourth embodiment of the present invention will be described.

(Example 1)
As a typical example of reverse micelle extraction performed in a conventional system, there are normal extraction and reverse extraction operations using cytochrome C which is a protein. Example 1 was conducted as a model experiment of continuous forward extraction and reverse extraction using the same extraction system as the conventional system in order to verify the effect of the micro extraction system (reverse micelle type microreactor) using reverse micelles. Is. The surfactant, organic solvent and protein used in Example 1 are as follows.

Surfactant: Sodium di (2-ethylhexyl) sulfosuccinate Organic solvent: Isooctane protein: Cytochrome C
As shown in FIG. 44, the positive extraction of Example 1 was carried out by flowing an isooctane solution containing 50 mM AOT at a predetermined flow rate from the sixth inlet 96 of the microreactor maintained at a constant temperature of 25 ° C. and from the seventh inlet 97. The two phases were brought into contact with each other by flowing a pH 8.5 aqueous solution containing 10 μM cytochrome C and 0.1 M potassium chloride at the same flow rate as that of the sixth inlet 96.

  The back extraction of Example 1 was carried out by adding 0.5 M of 50 mM AOT isooctane solution solubilized with cytochrome C obtained by the above-described normal extraction operation from the eighth inlet 98 to the sixth inlet 96 at the same flow rate. It was performed by flowing a potassium chloride aqueous solution into contact.

  The concentration of cytochrome C was measured using a spectrophotometer (wavelength 280 nm).

(result)
In Example 1, the following remarkable effects were obtained as compared with the conventional reverse micelle extraction.

  The micro extraction system using reverse micelles (reverse micelle type microreactor) is energy saving because it does not require stirring as an emulsification operation. In addition, since it was possible to avoid inactivation due to shear denaturation of the protein accompanying stirring, a highly active protein could be extracted. Since no phase separation operation is required, for example, no operation such as centrifugation is required, and no settling part for phase separation is required as in the conventional method, so the extraction time is significantly shortened, and the extraction process and operation Will also be simple.

  In the microextraction system (reverse micelle type microreactor) using reverse micelles as in Example 1, the operation and process of stirring and phase separation are omitted, and bio-related substances can be extracted in a short time. Efficiency (see FIG. 45) The normal extraction and the reverse extraction could be performed quickly and continuously.

(Example 2)
The surfactant, organic solvent and protein used in Example 2 are as follows.

Surfactant: Sodium di (2-ethylhexyl) sulfosuccinate Organic solvent: Isooctane Protein: Cytochrome C, lysozyme, Ribonuclease A
46, as shown in FIG. 46, an isooctane solution containing 50 mM AOT was allowed to flow from a sixth introduction port 96 of a microreactor maintained at a temperature of 25 ° C. at a predetermined flow rate. Positive extraction of cytochrome C and lysozyme is performed by flowing a pH8.5 aqueous solution containing 3 μM cytochrome C, 3 μM lysozyme, 3 μM ribonuclease A and 0.1 M potassium chloride at the same flow rate as the sixth inlet 96 and bringing the two phases into contact. It was. Liponuclease A was not positively extracted during the positive extraction process, and was recovered from the outlet of the raw material aqueous phase.

  The back extraction of Example 2 was performed according to the two-stage extraction conditions shown below.

  First, a 50 M AOT isooctane solution solubilized with cytochrome C and lysozyme obtained by the above-described positive extraction operation was supplied with a 0.5 M potassium chloride aqueous solution from the eighth inlet 98 at the same flow rate as the sixth inlet 96. Cytochrome C was back-extracted by contact.

  Next, a 50 M AOT isooctane solution solubilized with lysozyme obtained by the above positive extraction is brought into contact with a 1.5 M potassium chloride aqueous solution flowing from the 99th inlet 9 at the same flow rate as the sixth inlet 96. The lysozyme was back-extracted.

  Protein was quantified by high performance liquid chromatography.

  The measurement conditions for high performance liquid chromatography are as follows.

  Using ODS (0.30 mX4.0 mmφ) for the column, the flow phase is changed from 0.1% trifluoroacetic acid aqueous solution to 60% acetonitrile / 40% water / 0.1% trifluoroacetic acid for 15 minutes at a flow rate of 1 ml / min. did. An ultraviolet absorption detector (wavelength 280 nm) was used.

(result)
By the above-described forward extraction and two-step back extraction, the three proteins ribonuclease A, cytochrome C, and lysozyme could be separated and extracted at 100% (see FIG. 47).

(Example 3)
The surfactant, organic solvent and denatured protein used in Example 3 are as follows.

Surfactant: Sodium di (2-ethylhexyl) sulfosuccinate Organic solvent: Isooctane modified protein: Denatured ribonuclease A
48, as shown in FIG. 48, an isooctane solution containing 400 mM AOT was allowed to flow from a sixth introduction port 96 of a microreactor maintained at 20 ° C. at a predetermined flow rate, and a seventh introduction port 97 was used. Flow of 0.1 mg Tris buffer (pH 8.7) containing 2 mg / ml denatured ribonuclease A and 0.3 M guanidine hydrochloride at the same flow rate as that of the sixth inlet 96 and contact the two phases to correct the denatured ribonuclease A. Extraction was performed.

  Next, in the 400 mM AOT isooctane solution solubilized with the denatured ribonuclease A obtained by the above-described positive extraction operation, 0.1 M containing 0.1 M potassium chloride from the eighth inlet 98 at the same flow rate as that of the sixth inlet 96. The guanidine hydrochloride extracted together with the denatured clease A in reverse micelles was removed by back extraction by flowing Tris buffer (pH 8.7) into contact.

  Next, in the 400 mM AOT isooctane solution solubilized with the denatured ribonuclease A obtained by the above-described positive extraction operation, 0.6 mM reduced glutathione, 0.2 mM oxidized glutathione and 0.1 M are added at a predetermined flow rate from the tenth inlet 104. A 400 mM AOT isooctane solution containing Tris buffer (pH 8.7) was flowed to perform refolding. In this case, reduced glutathione, oxidized glutathione, and 0.1 M Tris buffer (pH 8.7) are incorporated into the reverse micelle water pool. In this process, the denatured ribonuclease was refolded.

  Next, 0.1 M containing 1 M potassium chloride and 100 μl / ml ethyl acetate is added to the 400 mM AOT isooctane solution solubilized with the active ribonuclease A obtained by the above refolding operation at a predetermined flow rate from the fifth inlet 25. Back extraction was performed by flowing and contacting with Tris buffer (pH 8.7).

  The concentration of ribonuclease A was measured using a spectrophotometer at a wavelength of 278 nm.

(result)
80% refolding of the denatured ribonuclease A was performed through a series of extraction operations and refolding steps using the above-described one chip.

(Fifth embodiment)
In the fifth embodiment, a system that performs biomimetic synthesis of protein using a microreactor including a molecular film will be described.

  As shown in FIG. 49, the cell-free protein synthesis process in the fifth embodiment includes a step of generating a cell-free solution (S1), a step of synthesizing a cell-free protein (S2), protein extraction and refolding. Step (S3) to perform, Step (S4) to regenerate ATP (adenosine triphosphate). Each step will be described below.

  (A) In step S1, as shown in FIG. 50, cell fluid is generated by membrane fusion. When a direct high voltage or alternating voltage is applied instantaneously to the electrodes 54a and 54b, the molecular film is disturbed and cell fusion is likely to occur. When the cell 8 is fused with the molecular film 5, the cell fluid contained in the cell is released in the same manner as in vivo cell fusion. Alternatively, membrane fusion may be performed by artificially changing the fluidity of the membrane by artificially controlling the temperature and pH conditions of the microchannel.

  (B) In step S2, as shown in FIG. 51, the hydrophobic membrane protein 103 synthesized by the protein biomimetic configuration system adheres to, deposits, permeates, or passes through the membrane by hydrophobic interaction with the molecular membrane 5. It can be incorporated into lipid bilayers. Therefore, the protein biomimetic composition system can prevent aggregation of membrane proteins biosynthesized by a normal protein biomimetic composition system or a cultured cell expression system, and synthesizes membrane proteins with high efficiency, in large quantities, and easily. be able to.

  In addition, by embedding ligands (for example, antibodies, receptors, substrates, glycolipids, etc.) capable of recognizing various proteins in the lipid bilayer membrane, the molecular membrane can specifically recognize the protein by bioaffinity.

  Furthermore, a protein can be selectively or specifically recognized by forming a membrane having a microdomain function partially or entirely in a macro channel.

  As described above, in step 2, using the electrostatic, hydrophobic, hydrophilic and ionic interactions between the membrane and protein, and bioaffinity, the protein biomimetic synthesis system of the present invention has high selectivity for various proteins. Highly efficient and can be synthesized in large quantities.

  In step S2, as shown in FIG. 52, a voltage or the like is applied to the electrodes 54a and 54b or the temperature is controlled, so that proteins and the like can permeate the molecular film 5. As the electrodes 54a and 54b, microelectrodes having an electrode surface such as a line shape or a dot shape can be used. According to this extraction method, peptides, polypeptides, proteins and the like synthesized by cell-free protein synthesis can be extracted with high activity. Further, since the produced protein is immediately extracted and separated, protein aggregation can be prevented.

  (C) Next, in step S3, the denatured protein produced by cell-free protein synthesis can be extracted and refolded. In particular, in step S3, the denatured protein is extracted and refolded using the microreactor having the molecular film of the first embodiment or the microextraction system (reverse micelle type microreactor) using the reverse micelle of the fourth embodiment. be able to.

  As an example of the refolding of the denatured protein, Example 3 of the microextraction system using reverse micelles according to the fourth embodiment can be cited, and detailed description of the embodiment is omitted here (FIG. 53).

(D) Next, in step S4, ATP (adenosine triphosphate) is regenerated. For example, FIG. 54 shows an example of regenerating ATP (adenosine triphosphate). Specifically, reduction of NAD ⁺ {nicotinamide adenine dinucleotide (oxidized form)} is first performed. NADH {nicotinamide adenine dinucleotide (reduced form)} is one of the energy sources required for the biosynthesis of ATP used as an energy source for cell-free protein synthesis from ADP in lipid bilayer membranes containing mitochondrial membrane components. is there. Since this NADH is consumed in the process of biosynthesis of ATP and converted to NAD ⁺ , it is first necessary to regenerate from NAD ⁺ to NADH. As shown in FIG. 54, a buffer solution of NAD ⁺ , ADP (adenosine diphosphate) and Pi (inorganic phosphate ion) is introduced from the first introduction port 22, and O ₂ is introduced from the second introduction port 23. Introduce. Here NAD ⁺ mediator modified electrode according to NAD ⁺ in the reduction performed, NADH is produced. Next, an amphipathic molecule mixed solution (lipid mixed solution 25) is introduced from the third introduction port 21, and a molecular membrane containing mitochondrion (lipid assembly membrane) or lipid bimolecular membrane is placed between the liquid / liquid laminar flow. Let it form. Then, ATP is generated by the oxidation and oxidative phosphorylation of NADH by mitochondria. Further, the reproduction of ATP in step S4 may be performed using the microchannel 3 having a shape as shown in FIG.

As shown in FIGS. 54 and 55, the reduction reaction of NAD ⁺ to NADH can be performed by the NAD ⁺ mediator modified electrode. On the other hand, NADH can also be regenerated from NAD ⁺ by a photoelectrochemical reduction method or an electrochemical reduction method in a microelectrolysis cell.

(E) FIGS. 56 to 58 show a method of regenerating NADH from NAD ⁺ by a photoelectrochemical reduction method or an electrochemical reduction method in a microelectrolysis cell in step S4.

Figure 56 illustrates a method of NADH in ATP synthesis is reproduce the optical electrochemically NADH to NAD ⁺ after being oxidized to NAD ^+. A molecular film (lipid bilayer) containing chlorophyll a, which is a photosensitive molecule, is formed between liquid / liquid laminar flows. Next, by irradiating the photosensitive membrane with light, chlorophyll a contained in the lipid bilayer membrane is activated by light energy, and electrons are released. NAD ^{+ in} channel 1 receives this electron and is reduced to NADH. On the other hand, chlorophyll a that has lost electrons receives electrons from Fe ²⁺ on the opposite side of the film, and Fe ²⁺ is oxidized to Fe ³⁺ . This Fe ³⁺ can be re-reduced to Fe ²⁺ by an electrochemical method, and is again supplied to the channel 2 from the second inlet 23 and reused.

FIGS. 57 and 58 show a method of regenerating NADH from NAD ⁺ using a microelectrolysis cell. Specifically, a method of reducing NAD ⁺ by electrochemical oxidation of water, which is partitioned between two liquid / liquid laminar flows by a molecular film (lipid bilayer film) having a function as a diaphragm.

FIG. 57 shows a method of regenerating NADH from NAD ⁺ by a molecular film (lipid bimolecular film) formed of uncharged lipid containing gramicidin serving as a diaphragm of an electrochemical cell.

In FIG. 58, a lipid bilayer or molecular membrane formed of uncharged lipid functions as a diaphragm of an electrochemical cell. By installing a saturated KCL salt bridge 57 in this system, K ⁺ and Cl ⁻ ions can be appropriately replenished to the fluid on both sides of the membrane, respectively. Using such a lipid bilayer membrane or molecular membrane type microelectrolysis cell, NADH is regenerated from NAD ⁺ .

The role of the diaphragm in the electrochemical reduction of NAD ⁺ is described in detail below. First, a constant constant voltage is applied by a potentiostat on the working electrode side (anode side) to cause an oxidation reaction of water.

H ₂ O − e ⁻ ⇔ 1/2 O ₂ + H ⁺
Simultaneously with the oxidation of water at the anode, the reduction of NAD + is carried out on the counter electrode side (cathode) as follows:

^{NAD + + e - + H +} ⇔ NADH
The oxygen produced at the working electrode is electrochemically very reactive. In other words, oxygen easily undergoes a reduction reaction and is electrolytically reduced to hydrogen peroxide or water.

Therefore, in the electrochemical reaction system as described above, when the working electrode and the counter electrode are present in the same microchannel system (having the same meaning as the same electrolysis cell) and without a diaphragm, oxygen generated at the working electrode is rapidly It is not desirable because it diffuses to the counter electrode side and interferes and affects the reduction reaction of NAD ⁺ . In particular, in the microchannel, the diffusion of the reaction product at each electrode is greatly accelerated, and the reaction product at the working electrode or the reaction product at the counter electrode interferes with the reaction at the relative electrode. Problems such as failure to obtain a reaction product occur.

  By providing a molecular film formed between both liquid / liquid laminar flows of the microchannel according to the fifth embodiment, in particular, a lipid bimolecular film, a laminar flow having a working electrode and a laminar flow having a counter electrode are partitioned. That is, both the working electrode chamber and the counter electrode chamber can be isolated.

  Also, the thickness of the lipid bilayer membrane provided in the microchannel is only 30-60 mm, and despite the fact that it is thin at the molecular level, the presence of this ultrathin membrane makes it possible to react with the reaction product in the conventional microreactor. Electrochemical reactions that could not be carried out efficiently due to the influence of interfering reactions due to diffusion can now be carried out efficiently.

  Furthermore, this ultrathin membrane is characterized in that it can selectively permeate by using a lipid bilayer membrane containing an ion channel, and has characteristics as an ion exchange resin type membrane.

As the ion channel, for example, gramicidin that selectively transmits hydrogen ions as shown in FIG. 57 can be used. In this system, gramicidin can immediately transport hydrogen ions generated by water oxidation on the anode side to the laminar flow on the reduction side of NAD ⁺ . Since the transported hydrogen ions are used for the reduction reaction of NAD ⁺ , the reduction reaction of NAD + can proceed smoothly.

(Operation and effect of the microreactor according to the fifth embodiment)
In the fifth embodiment, since intracellular substances are extracted by cell membrane fusion in the same manner as in biological systems, cell suicide caused by conventional cell crushing does not occur, and the activity of intracellular substances is reduced. Extraction can be performed while keeping. When the intracellular substance extracted by such a method is used for protein synthesis, high-efficiency synthesis similar to intracellular protein synthesis can be obtained.

  Further, the fifth embodiment is characterized in that a nucleic acid (RNA or DNA) virus in the virus and a protein called capsomer are extracted by membrane fusion between the virus and the membrane.

  In addition, the present invention can regenerate ATP, which is one of energy sources necessary for protein synthesis, by a series of electrochemical, photoelectrochemical, and chemical methods in a microreactor including a molecular film.

  Furthermore, protein synthesis using a microreactor system equipped with a molecular membrane can synthesize various proteins (genetically modified proteins, toxic proteins that cannot be synthesized in living cells) in large quantities and rapidly, as with cell-free protein synthesis. It is characterized by having properties that prevent denaturation due to protein aggregation due to the interaction between the synthesized protein and the molecular membrane, which is not in a cell-free system, that is, excellent live cell simulation properties.

  In the fifth embodiment, an electrostatic organ, hydrophobic, hydrophilic and ionic interaction between a cell and a molecular membrane, in particular, a lipid bilayer membrane, membrane fusion by bioaffinity, and a cell organelle (nucleic acid, Protein, endoplasmic reticulum, Golgi apparatus and ribosome).

  Hydrophobic membrane proteins synthesized by protein biomimetic construction systems are attached to or deposited on membranes by hydrophobic interaction with molecular membranes or lipid bilayer membranes, or are deposited in molecular membranes or lipid bilayer membranes. It is taken in.

  Accordingly, the protein biomimetic synthesis system of the present invention can prevent aggregation of membrane proteins biosynthesized in a normal cell-free protein synthesis system or cultured cell expression system, and synthesizes membrane proteins with high efficiency, in large quantities, and simply. It can be done.

  In addition, by embedding ligands (eg, antibodies, receptors, substrates, glycolipids, etc.) capable of recognizing various proteins in the lipid bilayer membrane, the lipid bilayer membrane can specifically recognize the protein by bioaffinity. . Furthermore, a protein can be selectively or specifically recognized by forming a membrane having a microdomain function partially or entirely in a macro channel. As described above, utilizing the electrostatic, hydrophobic, hydrophilic and ionic interactions between membranes and proteins, and bioaffinity, the biomimetic synthesis system for proteins of the present invention has high selectivity and high efficiency. And it is characterized by being able to synthesize | combine in large quantities.

  Molecular membranes and lipid bilayers in microchannels have characteristics as a diaphragm for electrochemical reactions. Molecular membranes formed between both liquid / liquid laminar flows of microchannels, especially lipid bilayer membranes have an electrochemical membrane function, and in particular lipid bilayer membranes containing ion channels selectively permeate ions. It has a characteristic as an ion exchange resin type diaphragm. Using such a microelectrolysis cell, NADH and ATP can be efficiently regenerated.

  By integrating and using several steps described above on one chip, it is possible to synthesize a protein with high efficiency, energy saving and continuous with one chip.

Example 1
Example 1 shows an example in which phospholipase, which is a hydrophobic protein, is synthesized using a microreactor equipped with a molecular film as shown in FIG. 51 in the second step of the protein biosynthesis system. The experimental conditions are as follows.

Cell extract: Escherichia coli S30 fraction extract (prepared from E. coli BL21 strain (Zubay, et al., Annu. Rev. Geneti., 7, 267 (1973)).
Plasmid DNA: Plasmid PLD (phospholipase) 12 (derived from Streptomyces antibioticus (Iwasaki, et al., J. Biosci. Bioeng., 89, 506 (2000)).
Protein synthesis solution: 50 mM Tris buffer (pH 7.4), 1.2 mM ATP, 1.0 mM GTP, 1.0 mM CTP, 1.0 mM UTP, 1.0 mM amino acid (20 L types), 2 mM dithiothreitol, 4% polyethylene glycol 6000, 35 μg / ml folinic acid, 0.17 mg / ml E. coli tRNA, 36 mM ammonium acetate, 8 mM magnesium acetate, 100 mM potassium acetate, 7.7 μg / ml T7 RNA polymerase, 25% E. coli S30 fraction extract, 15 μg / ml Plasmid PLD (phospholipase) 12
A protein synthesis solution is introduced at a predetermined flow rate from the second inlet 23 of the microreactor adjusted to a constant temperature condition of 37 ° C., and a 50 mM Tris buffer (pH 7.4) is introduced at a predetermined flow rate from the first inlet 22; An n-decane solution containing 5 mg / ml dioctadecyldimethylammonium bromide was introduced from the third inlet 21 at a predetermined flow rate. In this system, a hydrophobic protein called phospholipase was synthesized. By collecting the molecular membrane and lipid bilayer membrane formed between the liquid / liquid laminar flow at the outlet, a mixed solution of phospholipase and lipid was obtained. From this mixed solution, phospholipase can be separated by dialysis, or the activity of phospholipase can be measured by the following analytical method while the mixed solution is kept.

  Add phospholipase to a mixture of 8 μl of substrate solution (16 mM phosphatidyl-p-nitrophenol, 5% Triton X-100, 0.1 M Tris buffer (pH 8.0)) and 146 μl of 0.1 M sodium acetate (pH 5.5). A predetermined amount of the contained sample solution is added, and the progress of the reaction is followed at 25 ° C. and absorbance at 405 nm (characteristic absorption wavelength of p-nitrophenol).

(result)
The phospholipase synthesized by the protein biomimetic system using the microreactor equipped with the molecular membrane showed 20% higher activity than the phospholipase synthesized by the normal cell-free protein synthesis system.

(Other embodiments)
Although the present invention has been described according to the above-described embodiments, it should not be understood that the descriptions and drawings constituting a part of this disclosure limit the present invention. From this disclosure, various alternative embodiments, examples, and operational techniques will be apparent to those skilled in the art.

  For example, in the embodiment according to the present invention, specific examples such as proteins and peptides are given, but what can be used is not limited to those mentioned here.

  In the embodiment according to the present invention, it has been described that each solution is flowed in a laminar flow. It is also possible to stop the flow for measurement after flowing the solution.

    As described above, the present invention naturally includes various embodiments not described herein. Therefore, the technical scope of the present invention is defined only by the invention specifying matters according to the scope of claims reasonable from the above description.

It is a block block diagram of the analysis system which concerns on 1st Embodiment. It is a schematic diagram of the preparation part (microreactor) which produces the two-dimensional (planar) molecular film which concerns on 1st Embodiment (the 1). It is a schematic diagram of the preparation part (microreactor) which produces the two-dimensional (planar) molecular film which concerns on 1st Embodiment (the 2). It is a schematic diagram of the preparation part (microreactor) which produces the three-dimensional (cylindrical) molecular film which concerns on 1st Embodiment. It is a schematic diagram which shows the single tube | pipe type and multi-tube type microreactor which concern on 1st Embodiment. It is a schematic block diagram of the micro reactor analysis system (biosensing system) which concerns on 1st Embodiment (the 1). It is a schematic block diagram of the microreactor analysis system (biosensing system) which concerns on 1st Embodiment (the 2). It is a schematic block diagram of the micro reactor analysis system (biosensing system) which concerns on 1st Embodiment (the 3). It is the manufacturing method of the integrated film which concerns on 1st Embodiment (the 1). It is a manufacturing method of the integrated film which concerns on 1st Embodiment (the 2). It is a schematic process diagram of the bioreactor according to the first embodiment (No. 1). It is an example of the product produced | generated in the bioreactor of FIG. FIG. 2 is a schematic process diagram of the bioreactor according to the first embodiment (No. 2). It is an example of the product produced | generated in the bioreactor of FIG. FIG. 3 is a schematic process diagram of the bioreactor according to the first embodiment (No. 3). It is a schematic process figure of the bioreactor which concerns on 1st Embodiment (the 4). FIG. 5 is a schematic process diagram of the bioreactor according to the first embodiment (No. 5). FIG. 6 is a schematic process diagram of the bioreactor according to the first embodiment (No. 6). FIG. 7 is a schematic process diagram of the bioreactor according to the first embodiment (No. 7). It is the schematic of extraction of the intracellular organelle by the membrane fusion which concerns on 1st Embodiment. It is the schematic of extraction and isolation | separation of the bio-related substance by interaction with the film | membrane or film | membrane which concerns on 1st Embodiment. It is a block schematic diagram of the microreactor of Example 1 which concerns on 1st Embodiment. It is a graph which shows the time-dependent change of the conversion rate in the comparative example 1 and Example 1 which concern on 1st Embodiment. It is a block schematic diagram of the microreactor of Example 2 which concerns on 1st Embodiment. It is a graph which shows the relationship between the yield of N-acetyl-late tryptophan ethyl ester and flow rate ratio in Example 2 which concerns on 1st Embodiment. It is a block schematic diagram of the microreactor of Example 3 according to the first embodiment. It is a block schematic diagram of the microreactor of Example 6 in the first embodiment. It is the structure schematic of the microchannel sensor which concerns on 2nd Embodiment (the 1). It is the structure schematic of the microchannel sensor which concerns on 2nd Embodiment (the 2). It is the structure schematic of the microchannel sensor which concerns on 2nd Embodiment (the 3). It is the structure schematic of the microchannel sensor which concerns on 2nd Embodiment (the 4). It is the structure schematic of the microchannel sensor which concerns on 2nd Embodiment (the 5). It is the structure schematic of the microchannel sensor which concerns on 2nd Embodiment (the 6). It is the structure schematic of the microchannel sensor which concerns on 2nd Embodiment (the 7). It is the structure schematic of the microchannel sensor of the comparative example which concerns on 2nd Embodiment, and an Example. It is a conceptual diagram which shows the detection principle of the structurally abnormal protein which concerns on 3rd Embodiment. It is a block schematic diagram of the microreactor of Example 1 which concerns on 3rd Embodiment. It is a structure schematic of the microreactor of Example 2 which concerns on 3rd Embodiment. It is a block schematic diagram of the microreactor of Example 4 which concerns on 3rd Embodiment. It is a block block diagram of the micro reactor system which concerns on 4th Embodiment. It is the structure schematic of the microreactor in the normal extraction part of FIG. 40 (the 1). It is the structure schematic of the microreactor in the normal extraction part of FIG. 40 (the 2). It is the structure schematic of the microreactor in the back extraction part of FIG. It is a block schematic diagram of the microreactor of Example 1 which concerns on 4th Embodiment. It is a table | surface which shows the result of Example 1 which concerns on 4th Embodiment. It is a block schematic diagram of the microreactor of Example 2 which concerns on 4th Embodiment. It is a table | surface which shows the result of Example 2 which concerns on 4th Embodiment. It is a structure schematic of the microreactor of Example 3 which concerns on 4th Embodiment. It is a block block diagram of the protein biological simulation synthesis system which concerns on 5th Embodiment. It is the structure schematic of the microreactor in step S1 of FIG. FIG. 50 is a schematic configuration diagram of a microreactor in step S2 of FIG. 49 (No. 1). FIG. 50 is a schematic configuration diagram of a microreactor in step S2 of FIG. 49 (part 2). It is the structure schematic of the microreactor in step S3 of FIG. It is the structure schematic of the microreactor in step S4 of FIG. 49 (the 1). FIG. 50 is a schematic configuration diagram of a microreactor in step S4 of FIG. 49 (part 2). FIG. 50 is a schematic configuration diagram of a microreactor in step S4 of FIG. 49 (No. 3). FIG. 50 is a schematic configuration diagram of a microreactor in step S4 of FIG. 49 (No. 4). FIG. 50 is a schematic configuration diagram of a microreactor in step S4 of FIG. 49 (No. 5).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microreactor 2 Reverse micelle type microreactor 3 Microchannel 5 Molecular film 6 Monomolecular film 7 Integrated film 8 Cell 9 Bio-related substance 11 Production / regeneration part 12 Condition control part 13 Detection part 14 Reaction part 15 Circulation part 16 Separation / recovery part 21 First inlet 22 Second inlet 23 Third inlet 24 Fourth inlet 25 Fifth inlet 26 Second fluid 27 Third fluid 28 Amphiphilic molecule mixture 31 Aqueous phase 32 Water phase 33 Lipid phase 34 Molecular membrane layer (lipid assembly membrane layer)
35 Cylindrical lipid bilayer membranes 40a, 40b,..., 40f Barrier 41 Filter 42 Structure abnormal protein 43 Structure abnormal protein aggregate 44 Amyloid protein 45 Amyloid protein aggregate 46 Membrane potential measuring device 51a, 51b Electrode 52 Electricity Chemical signal measuring device 53a, 53b Electrode 54a, 54b Electrode 55 Cathode 56 Anode 57 Salt bridge 60 HIV-1
61 Fluid 62 Insulating medium 63 Fluid 64 Amphiphilic molecule mixture (molecular film forming solution)
65 Fluid or Fluid Containing Sample 66 Insulating Medium 67 Fluid 71 Electrochemical Measuring Device 72a, 72b Electrode 73 Sensor Electrode 74 Reference Electrode 75 Counter Electrode 76 Ion Channel 77 Salt Bridge 78 Insulating Medium 79 Molecular Membrane (Lipid Assembly Membrane)
80 Aqueous solution 81 Aqueous solution containing HIV 91 Positive extraction unit 92 Raw material recovery unit 93 Back extraction unit 94 Product recovery unit 95 Circulation unit 96 Sixth introduction port 97 Seventh introduction port 98 Eighth introduction port 99 Ninth introduction Mouth 100 Inlet 101 Raw material aqueous phase 102 Recovered aqueous phase 103 Synthesized membrane protein 104 Tenth inlet 105 Eleventh inlet 106 Twelve inlet

Claims (12)

A microchannel, a sixth inlet for introducing a reverse micelle solution into the microchannel, a seventh inlet for introducing a raw material aqueous solution containing a substance into the microchannel, a redox agent, a molecular chaperone, water, a metal A tenth inlet for introducing a reverse micelle solution containing ions, and in the microreactor, a substance in the raw water phase is positively extracted into the reverse micelle solution, or refolding of a denatured protein accompanying extraction is performed. A positive extraction unit to be performed;
A raw material recovery unit for recovering or recirculating the raw material aqueous solution;
A microchannel, an eleventh inlet for introducing the reverse-extracted reverse micelle solution into the microchannel, and a twelfth inlet for introducing the recovered aqueous solution into the microchannel; A reverse extraction unit that performs reverse extraction of a substance or refolded protein from the reverse micelle solution,
A circulating part for circulating the reverse micelle solution;
An extraction system comprising: a product recovery unit that recovers the back-extracted substance or the refolded protein.   The reverse micelle is a reverse micelle composed of at least one kind of amphiphilic molecule, at least one kind of amphiphilic molecule and at least one kind of co-surfactant, ligand, phospholipid, glycolipid, cholesterol, protein. The extraction system according to claim 1, wherein the extraction system is a mixed reverse micelle comprising a sphingolipid, a redox species, a cell recognition molecule, and a receptor.   The nucleic acid-related substance and / or protein are mixed in the water pool present in the center of the reverse micelle, or these are mixed, and the biological substance is incorporated therein. Extraction system.   A redox agent, a molecular chaperone, and a metal ion are incorporated into a reverse micelle water pool containing the denatured protein, and the three-dimensional structure and activity of the protein are restored in the reverse micelle water pool. 2. The extraction system according to 1.   The back extraction unit includes a branch channel for controlling the pH or salt concentration of the recovered aqueous phase or a branch channel for adding a hydrophilic organic solvent, and further includes a device for controlling the temperature and pressure conditions of the microchannel. The extraction system according to claim 1. A microchannel, a sixth inlet for introducing a reverse micelle solution into the microchannel, a seventh inlet for introducing a raw material aqueous solution containing a substance into the microchannel, a redox agent, a molecular chaperone, water, a metal A tenth inlet for introducing a reverse micelle solution containing ions, and in the microreactor, a substance in the raw water phase is positively extracted into the reverse micelle solution, or refolding of a denatured protein accompanying extraction is performed. The steps to be taken,
Collecting or recirculating the raw material aqueous solution;
A microchannel, an eleventh inlet for introducing the reverse-extracted reverse micelle solution into the microchannel, and a twelfth inlet for introducing the recovered aqueous solution into the microchannel; Back-extracting the substance or refolded protein from the reverse micelle solution,
Circulating the reverse micelle solution;
Recovering the back-extracted substance or the refolded protein. A cell supply system for supplying cells;
A cell-free solution generating system that generates cell-free solution;
An amino acid supply system for supplying amino acids;
A nucleic acid supply system for supplying nucleic acid;
An energy supply system for supplying energy;
A cell-free protein synthesis system for synthesizing proteins;
An extraction / refolding system for extracting or refolding the protein synthesized in the cell-free protein synthesis system using the extraction system according to claim 1;
An ATP regeneration system that recovers and regenerates ATP from the cell-free protein synthesis system or from the extraction / refolding system;
A protein synthesis system comprising: a protein recovery system that recovers a protein from the cell-free protein synthesis system or from the extraction / refolding system.   The cell-free fluid production system is a microreactor equipped with a molecular membrane, by electrostatic, hydrophobic, hydrophilic and ionic interactions between cells and molecular membranes, particularly lipid bilayer membranes, membrane fusion by bioaffinity, Alternatively, the protein synthesis system according to claim 7, wherein organelles and cytoplasmic substrates in the cell are extracted by applying a stimulus to the molecular membrane.   The cell-free protein synthesis system synthesizes a protein in a microreactor equipped with a molecular membrane, and the synthesized protein is subjected to electrostatic, hydrophobic, hydrophilic and ionic interactions with the molecular membrane, bioaffinity, 8. The protein synthesis system according to claim 7, wherein the protein synthesis system avoids protein denaturation due to protein aggregation by attaching to, adsorbing, depositing, permeating or extracting by being incorporated into a molecular film.   8. The protein synthesis system according to claim 7, wherein the protein extraction / refolding system performs protein extraction and refolding using a microreactor having a molecular film.   The protein synthesis system according to claim 7, wherein the ATP regeneration system regenerates ATP by electrochemical reaction, photoelectrochemical reaction, and chemical reaction in a microreactor having a molecular film. Supplying a cell-free solution from the cell supply system to the cell-free solution generation system, and extracting and generating the cell-free solution in the cell-free generation system;
Cell-free protein synthesis system supplies cell-free protein from cell-free fluid generation system, amino acid from amino acid supply system, nucleic acid from nucleic acid supply system, ATP from ATP generation system, energy by energy supply system Synthesizing a protein in a synthesis system;
Extracting and refolding the protein synthesized by the cell-free protein synthesis system using the extraction system according to claim 1;
Collecting and regenerating ATP from the extraction / refolding system;
Recovering the protein produced by the extraction / refolding system.