JP2011013208A - Biological operation system and industrial operation system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a labo-on-the-disk (chip) type biological operation system acquiring accurate information, while usable easily, and to provide an industrial operation system separating and detecting a true sphere as a standard sphere.SOLUTION: In the biological operation system, one carrier includes a body fluid-based solution adjustment input part for adjusting and inputting a body fluid-based solution including cells or a medium including spheres, a separation extraction part for separating and extracting objective particles or liquid relative to particles and liquid in adjustment liquid inputted into the adjustment input part, and an analysis operation means for performing analysis operation of the particles or the liquid acquired by the separation extraction part, and the means of the carrier is connected through a channel, and the channel performs moving operation of the body fluid-based solution by a flow generated by a physical driving means. The industrial operation system is also provided.

Description

  TECHNICAL FIELD The present invention relates to a biological operation system used for immunodiagnosis, health / disease diagnosis, infectious disease test, antibody production, and the like, and a body fluid system using a micro-channel such as a lab on the desk (chip). The present invention relates to a system using a device for operation-intensive processing of a solution and an industrial operation system for discriminating and separating a true sphere of a specific size used as a standard body of a measuring machine instrument.

A system that can diagnose lifestyle-related diseases at home by increasing the sensitivity of reagents, elucidating antigen-antibody reactions, and examining body fluid components has recently attracted attention because of the rationalization of the medical field.
As for the collection of body fluids used for diagnosis of various diseases and immunodiagnosis, there is still no change in overall collection using an injection needle due to its quantitativeness and certainty. It is still mainstream.
The use of injection needles causes temporary pain to the living body, and in order to resolve the pain in a short period of time, there is a need for an accurate and reliable diagnosis with a small amount of body fluid depending on the situation. .
On the other hand, advances in microfabrication technology, advancements in computers, etc. enable the use of micro devices such as microchannels, microvalves, etc. for performing movement operations and classification operations in units of particles, The realization of particle-level operation units suggests the possibility of expansion of mobile diagnostics such as POC, and electrical equipment manufacturers that manufacture IC chips are actively expanding into this field. Sex has come to be seen.
This diversity includes, for example, DNA obtained from cells obtained by detection, so that useful things are separated from this DNA, amplified, and subjected to genetic manipulation used for treatment and testing. It is.

In other words, there are more proposals for chip-like devices that can be operated at the research and development level while having a smaller size, such as lab-on-the-desk (lab-on-the-chip). . Japanese Patent Laid-Open Nos. 2000-180452, 2001-50384, 2009-63590, and the like have a configuration for performing fluid movement stop control using a rotational force, and disclose that the usage is diverse.
Patent Documents and Non-Patent Documents disclose means for achieving movement and classification in particle units, and Patent Document 5 supplies blood to the center of a rotor, and after removal and removal of blood cells by dilution and centrifugation It is disclosed that processes such as quantitative distribution of plasma, quantitative supply to the reagent section, and color development reaction are performed by adjusting the physical phenomenon of the capillary channel and the rotational motion.
A blood analyzer utilizing such centrifugal force has been proposed since the 1970s, and even in Japan, the blood force and capillary phenomenon as described in JP 2009-58354 A have been proposed. A diagnosis unit to be used has been proposed.
Non-Patent Documents 1 and 2 describe laminar flow, particle classification using T-shaped, cross-shaped main flow path and branch flow path, separation / concentration process, without using centrifugal drive, Proposals have also been made to obtain the particle size from the relationship between the main channel and the branch channel.
Japanese Patent Application Laid-Open No. 2009-511001 discloses that blood cells are separated by a magnetic field based on the magnetic properties of cells, and in particular, a method for separating nucleated red blood cells is described.

Further, JP-A-2009-511001 discloses that magnetic particles are further antibodies (for example, anti-CD71, anti-CD36, anti-CD45, anti-GPA, anti-antigen i, anti-CD34, It is described that the sensitivity to magnetic force is further increased by including fetal hemoglobin, anti-EpCAM, anti-E-cadherin, or anti-Muc-1) or an antigen-binding fragment thereof.
Japanese Patent Application Laid-Open No. 2004-77387 discloses a first antibody reaction part containing an anti-human myoglobin rabbit antibody labeled with colloidal gold with a labeling kit and a second antibody reaction part having an anti-rabbit IgG mouse antibody immobilized on a microchannel. And an immune chip for detecting human myoglobulin in blood is shown.
In Japanese Patent Publication No. 8-1434, thiazole orange, a fluorescently labeled anti-CD45 monoclonal antibody and a fluorescently labeled anti-CD71 monoclonal antibody are added to a blood sample, and then cells stained or bound with a fluorescent label via the antibody are flow sites. It is described that it is detected by a meter.
These cited examples show a configuration for detection by staining of target cells in blood, components, and antigen-antibody reaction.

JP 2007-21465 JP 2007-175684 A JP-A-7-60193 JP 2004-88843 A Special table hei 5-508709 JP 2009-058354 A JP 2009-511001 JP 2004-77387 A Japanese Patent Publication No.8-1434

M.M. Yamada, M .; Seki, Biomed Microdevice (2007) 9: 637-645 M.M. Yamada, M .; Seki, LabChip, 2005, 5, 1233-1239

  It is difficult to perform a series of operations such as blood cell separation, quantitative operation, reagent color reaction, etc. by rotating a rotor equipped with a microchannel, centrifuge, reagent reaction unit, etc., simply by rotating the rotor. Therefore, it is necessary to perform highly accurate rotation, change of rotation direction, stop, etc., and so-called encoding-controlled rotation is required, which makes the apparatus complicated and expensive. Furthermore, giving an environmental change that is significantly different from the original environment of the biological sample, such as strong centrifugal force due to high rotation, often affects the properties of the biological sample itself, making it impossible to obtain appropriate test results and separation results. The possibility is also a concern.

Since the operation in particle units is an operation in a minute region, the components for driving are also miniaturized, so it receives a force such as capillary force that is different from that of a normal device. In this case, more conditions must be overcome, and it is required to realize a diagnostic device by manipulating body fluid with a simpler configuration.
In addition, these micro-channels, valves, quantitative operations, and classification operations that have been developed over the past few decades are simpler and easier to handle fluid handling chips that are applied to practical applications. Does not yet exist. The reason why it is difficult to put to practical use is that it is very difficult to mass-produce chips at a micrometer level with high accuracy. For example, if a cell having a size of several micrometers is to be realized only by a micro structure, structural accuracy management in units of 1 micrometer is required. Especially when mass production is premised, resin molding with a mold is a typical method, but in order to smoothly remove the molded part from the mold, it is necessary to give a gradient to the vertical wall part as well. A micrometer-level structure will have a significant impact on physical properties. In addition, the surface tension of the liquid has a great influence in the fine structure. For example, when bubbles are mixed, it does not move easily, and can also be a factor for changing physical properties. Since a normal operation cannot be performed by a human under a vacuum condition, mixing of bubbles is a big problem in practical use.

In view of the above, the present invention
A component adjustment input unit that adjusts and inputs biological components such as body fluid solutions,
A separation / extraction unit for separating and extracting a target component or liquid in the adjustment liquid adjusted and input to the component adjustment input unit, for analyzing and operating particles or liquid obtained in the separation / extraction unit and outputting related information One carrier provided with an analysis operation means, wherein each means of the carrier is connected by a flow path, and the flow path has a combined configuration in which the body fluid solution is moved and operated by a flow generated by a physical drive means. By moving the fluid, preferably in a unidirectional flow by laminar flow, and by discriminating unnecessary particles and securing the necessary liquid by the flow and the shape of the flow path, the reagent and test kit in the subsequent stage A configuration for providing target particles and liquids is realized. The component adjustment input unit does not necessarily perform all adjustments necessary for the use of the chip. For example, adjustments such as diluting blood or modifying fluorescent beads in advance to specific components are performed by the user outside the chip. Once done, the specimen may be introduced into the chip.
Based on the above configuration, the present invention also makes it possible to sort cells having similar sizes, such as nucleated red blood cells and white blood cells, using one or more carriers. That is, in the combination of branch channels connected to the main channel, blood is driven in a laminar flow to flow red blood cells to the branch channel, and the main channel is made only of blood cells of a white blood cell level, and then nucleated red blood cells or red blood cells Separation in a single flow, such as a structure that distinguishes based on particle size by means of separation and extraction combined with an immunological technique by binding to an antibody connected with a label for cell membrane antigen. A method is formed so that cells of the same size can be separated. This separation is equivalent to the detection of rare cells such as nucleated red blood cells that can be diagnosed with leukemia due to their presence, for example in adults. Make it possible. The rare cells shown in the present invention include mature reticulocytes, immature reticulocytes, mature nucleated red blood cells, immature reticulocytes, mature nucleated red blood cells, immature nucleated red blood cells, blast cells, plasma cells, megakaryocytes, neutrophils. Also included are spheres, eosinophils, basophils, monocytes, lymphocytes, and the like.

  In addition, the term “laminar flow” in this specification means a flow in which a turbulent flow or a vortex flow is not generated in a direction different from the direction suitable for the original purpose, and a plurality of flows from different inlets. It does not necessarily mean a general laminar flow phenomenon in which an interface is formed between the two layers and proceeds without crossing each other. The purpose of the system of the present invention is the separation and extraction of particles contained in the fluid, which is realized by separating a part of the fluid traveling in the main flow path to the branch flow path side. In other words, at the branch point between the main flow path and the branch flow path, it is divided into a flow that goes straight through the main flow path and a flow that changes direction toward the branch flow path, and these different flows travel along the same main flow path. For the sake of convenience, it may be expressed as “laminar flow”. The ratio of the flow rate of the flow that goes straight through the main flow path and the flow absorbed by the branch flow path side is strictly determined by the ratio of the resistance values of the respective flow paths. This means that a fluid component having a certain width travels to the branch channel side. It is convenient to express the flow toward the branch flow path having a certain width as “laminar flow on the branch flow path” and the flow straight on the main flow path as “laminar flow on the main flow path”. The greatest feature of the particle separation in the fluid by the system of the present invention depends on whether or not the particle (the position of the center of gravity) is included in the laminar flow width on the side of the branch flow path strictly determined by the ratio of the flow path resistance values. It is determined whether or not the particles are separated to the branch flow path side. After a part of the laminar flow on the branch channel side is absorbed by the branch channel, a part of the laminar flow on the main channel side is supplemented to the branch channel side, and the same separation phenomenon occurs at the next branch point. Therefore, these laminar flows do not intersect with each other, and are always in a complementary relationship. This has a different meaning from the general definition of laminar flow.

Biological components such as bodily fluid solutions in the present invention are obtained from a living body such as sweat, blood, semen, bone marrow fluid, pleural effusion, exudate, lymph, urine, feces, cerebrospinal fluid, saliva, and periodontal exudate. , Pesticide testing, food poisoning adjustment solution, rain water, water and sewage water, etc., and also contains cells such as cell suspension, or contains nucleic acids, proteins, cell contents, bacteria, microorganisms, viruses Solutions are also included.
In order to adjust the amount of the solution according to its viscosity and the size and shape of the flow path, a step of adjusting by adding a diluting solution such as physiological saline is included.
The separation and extraction unit according to the present invention performs, for example, physical drive, liquid filled in a syringe, drive by a pressurized pump via air, or volume change of a container containing liquid by displacement of a piezoelectric body. The flow of the fluid preferably forms a unidirectional flow in the fluid to carry the particles, and a branch channel is connected to the side of the main channel, and the width of the laminar flow entering the branch channel is set to the resistance of each channel. Controlled by the ratio of values, particle mixing does not occur. For example, in the case of blood, liquid components are extracted, and cells are captured and separated by external magnetic force or electric force, preferably fluid mechanics. In particular, a configuration in which a specific cell is supplemented by branching and moving in a branch channel is exemplified.

Unlike a general filter that separates particles according to the size relationship between the size of the structure such as a mesh and the size of the particle, the size of the structure is not always the same in the method of separating the particles according to the laminar flow width of the present invention. It is not necessary to be below the threshold value of the particle size to be separated. For example, even if the width of the entrance of the branch channel is 20 μm, if the width of the laminar flow on the side of the branch channel is designed to be 1 μm, 2 μm particles cannot enter the branch channel of 20 μm width. . This is a very significant feature of the present invention, and it is possible to separate a group of particles having a very fine difference of several μm using a physical structure that is relatively easy to manufacture of about several tens of μm. This eliminates the need for accuracy management at the level of several micrometers as described above as the first problem, and can dramatically improve chip productivity. The characteristics of the flow path configuration used in the present invention are as follows.
1. Having a structure in which there is a branch channel connected to the side of the main channel, and determining the width of the flow proceeding to the branch channel by controlling each flow rate proceeding to the main channel and the branch channel,
2. The means for controlling each flow rate proceeding to the main channel and the branch channel is based on the ratio of the channel resistance values of the main channel and the branch channel,
3. By making the width of the flow proceeding to the branch flow channel side smaller than the width of the branch flow channel, the separation threshold based on the particle size can be set smaller than the width of the branch flow channel,
The width of the flow traveling toward the branch channel is not more than half the width of the branch channel;

  In addition, it is more preferable that the number of branch flow paths connected to the main flow path is more than one. If there are a large number of particles of the same size included in the laminar flow on the branch flow path side, particles that can enter the laminar flow on the branch flow path side cannot enter due to interference between the particles, Since a phenomenon that is not separated on the branch flow path side occurs, by preparing a plurality of branch flow paths having the same size separation threshold, it is possible to reliably advance the particles to the designed branch flow path. This is a content that is determined probabilistically. For example, for a number n of particles existing in 1 ml, a branch flow path for separating the particles is in a range of logn to 20 logn (log is a logarithm with 10 as the base). It only has to exist.

The analysis operation means in the present invention includes, for example, an immunochromatographic test strip and a color development indicator reagent in addition to a method of obtaining a signal such as fluorescence or current value by directly fixing an antibody, a nucleic acid or the like on a flow path surface or a branch destination reservoir. Examples include using a test piece, a sensor, a counter, a spectrophotometer, a camera, an image sensor, a photodiode, a laser beam, or the like alone or in combination. When capturing particles using antigen-antibody reaction or nucleic acid hybridization, fix the antibody, nucleic acid, etc. so that the target particle is captured in the liquid reservoir, or other than the intended purpose in the liquid reservoir. There are two systems in which antibodies, nucleic acids and the like are fixed so as to capture the particles, and only the target particles are recovered from the downstream outlet portion. In addition, the size of each particle collected from the outlet can be changed by not allowing any particles to be captured inside the chip, but allowing the antibody, nucleic acid, etc., to which beads are attached, to adhere only to the specific particles of the sample after separation. However, size separation may be performed again. Thus, when using the principle of separation by particle size, particles are separated based on the composite particle size obtained by artificially binding the modified product to the particles, not the original particle size itself. It is also possible to perform separation based on the size of the complex (for example, a cell mass) or separation based on the length of the complex of chained particles. An appropriate flow path pattern may be designed and applied. These information includes, for example, color information, movement position information, fluorescence information, size information, other shape information, sequence information, state information such as the presence or absence of binding, immunoprecipitation information generated in the state of binding to beads, sedimentation Presence / absence information, presence / absence of charge, strength / polarity information, magnetic presence / absence / strength / polarity information, concentration information, temperature information, strain information, viscosity information, etc. Thus, it is also provided for a system for receiving lifestyle guidance, early detection of lifestyle-related diseases, and improvement guidance. Details are shown in Table 1.
Table 1 will be described.

Sample: Blood Separation target: Plasma Application field: clinical test Example 1: Measurement of glucose, lactic acid, creatinine, etc. by enzyme electrode method Example 2: Measurement of Na ⁺ , K ⁺ , Cl ⁻ by ion selective electrode method Example 3 : Measurement of pH by hydrogen ion selective electrode method Example 4: Measurement of blood oxygen concentration by Clark type electrode method Example 5: Immunoantibody reaction (ELISA, latex immunoturbidimetric method, colloidal gold aggregation method, octalony method, etc.) ) Measurement of various plasma protein antigens (cytokines, albumin, collagen, CRP, etc.) or blood antibodies (IgA, IgD, IgE, IgG, IgM, antiviral antibodies, antibacterial antibodies) Sample: Blood Separation target: Lymphocytes Field of application: clinical test Example 1: Measure T cell, B cell, NK cell activity Example 2: Analysis of cell surface markers (CD antigen) and cytokines produced (Th1 / Th2 etc.) Sample: Blood Separation target: Red Application area: clinical test Example 1: Separation and recovery of nucleated red blood cells derived from fetus from maternal blood Example 2: Separation sample according to difference in deformability of red blood cells: Blood Separation target: Circulating tumor cells Application field: Clinical examination Example 1: Determination of cancer metastasis

Sample: Bone marrow / blood Separation target: Hematopoietic stem cells Application field: clinical, experiment Example 1: Separation and concentration of hematopoietic stem cells and mesenchymal stem cells used for transplantation, experiments, etc. Since it is larger than other blood cells, it can be separated with HDF, and its presence is very low (0.004%), so concentration is useful.

Sample: Tissue Separation target: Target cell Application field: Experiment Example 1: Separation and concentration of parenchymal cells and non-parenchymal cells from liver tissue using the difference in size (Yamada, 2007)
Sample: Tissue Separation target: Stem cells Application field: Clinical, experiment Example 1: Isolation and concentration of tissue stem cells used for transplantation, experiments, etc. Since it is different in size from other cells, it can be separated by HDF, and its concentration is useful because it is not frequently present.
Sample: Tissue Separation target: iPS cells (artificial pluripotent stem cells)
Field of application: Regenerative medicine, etc. Example 1: Separate and collect only iPS cells from cells collected from tissues.
Example 2: Only cells differentiated into specific cells from the iPS cell group are separated and recovered.
Sample: Cultured cell Separation target: Synchronous cell Application field: Experiment Example 1: Separation and concentration using the difference in size for each cell cycle (G0 / G1, S, G2 / M phase)

Sample: Cultured cells Separation target: Apoptotic cells Application field: Experiment Example 1: Separation / concentration / removal using the fact that cells in apoptotic phase become smaller Sample: Cultured cell Separation target: Cell dead body Application field: Experiment Example 1 : Removal of cell dead bodies that hinder the experiment

Sample: Yeast Separation target: Synchronized cells Field of application: Experiment Example 1: Separation / concentration using the difference in size for each cell cycle (G0 / G1, S, G2 / M phase) Sample: Bacteria Separation target: Target Bacteria Field of application: Experiment, inspection Example 1: Separation / concentration using size differences at each division stage

Sample: Drinking water, sewage, environmental water Separation target: Fungus, bacteria, suspended particulate matter Application field: Water quality inspection, hygiene management Example 1: Pollution degree measurement / inspection sample: Drinking water, sewage, environmental water Separation target: pathogen Application areas: Water quality inspection, hygiene management Example 1: Detection of Cryptosporidium oocysts (pathogenic protozoa). concentrated

なお、数ナノメートル単位で微細構造を形成させることが技術的に可能となった現在では、流路形成時に検出対象を検出するための微細構造を同時に形成することにより、とくに流路形成後に検出のための分子等を定着あるいは付与する行程を不要とすることも可能である。例えば検出したいタンパク質あるいは核酸、細胞等と特異的に結合する構造を、抗体あるいは核酸を後から流路に定着させるのではなく、流路構造の一部として直接形成するといった方法が例示される。
その他、分析を行う対象として
抗原抗体反応による免疫計測、細菌、白血球の測定による感染症等の各種疾病、コ
レステロール、血糖等の成分値の測定、その他、表１参照 が例示される。

In addition, now that it is technically possible to form a fine structure in units of several nanometers, it can be detected especially after the flow path is formed by simultaneously forming a fine structure to detect the detection target when the flow path is formed. It is possible to eliminate the process of fixing or imparting molecules for the purpose. For example, a structure that specifically binds to a protein, nucleic acid, cell, or the like to be detected is directly formed as a part of the flow channel structure instead of fixing the antibody or nucleic acid to the flow channel later.
In addition, examples of the subject to be analyzed include immunoassay by antigen-antibody reaction, various diseases such as infectious diseases by measurement of bacteria and leukocytes, measurement of component values such as cholesterol and blood glucose, and others, see Table 1.

The present invention includes, for example, a sample separation unit having a liquid component separation / extraction unit formed by a branch channel extending in a direction perpendicular to or oblique to the main channel, or both of the main channel and the main channel. A free antibody placement part in which a labeled free antibody to be measured is placed in a branched flow path part that has been separated and extracted from a liquid component, and a fixed part in which the fixed antibody is placed behind the free antibody placement part. The combination of integrated carriers forming a measuring part for measuring the concentration and number of labels is shown.
Furthermore, the combination of the main flow channel and the branch flow channel enables the discrimination of particles having different particle sizes, but the present invention further has the same size but different properties that cannot be separated by the size of the branch flow channel. For example, the separation of nucleated red blood cells and white blood cells is indicated by one or more carriers provided with a separation unit using an antibody for red blood cells.
That is, the main channel or branch channel, which is branched from the main channel perpendicularly or obliquely, has a size that branches and removes mature (nuclear) red blood cells, and a branch channel. An antibody that specifically binds to red blood cells, and stores a free antibody that is bound to a label formed of colloidal particles made of gold, platinum, a magnet component, a fluorescent component, and the like, and the antibody in the subsequent stage. By forming a fixed part in which the fixed membrane is arranged, nucleated red blood cells can be captured and detected or detected from particles of approximately the same size after separating the mature red blood cells.
As a detection method, a surface plasmon resonance mass spectrometer or other analyzer according to the label may be used.

In the case of nucleated red blood cells, the number of detections detected from the peripheral blood of adults and the presence thereof are related to various diseases such as anemia and leukemia. Therefore, the above-described combination is an example of immunodiagnosis.
The antibody that binds to the cell membrane surface antigen of nucleated erythrocytes may be the same as the antibody against the cell membrane antigen of erythrocytes. For example, Glycophorin A (BD Bioscience Pharmingen), Band 3, Anion Exchanger 1 (Santa Cruz), common Leukocyte Antigen (BD Bioscience Pharmingen), glycosyl-phosphatidylinositol (GPI) -linked single chain membrane glycoprotein, Epsilon-globlin (Europa Bioproducts, UK), Transferrin Receptor (BD Bioscience Pharmingen), Integrin-Associated protein (BD Bioscience Pharmingen), Thrombospondin receptor, Complement receptor 1 (BD Bioscience Pharmingen) are shown. In addition, the combination for detecting nucleated red blood cells is not limited to this. For example, a free antibody in which only the fixing part to which the above antibody is fixed is used, and after passing, the colloidal particles having gold, platinum and magnetism are bound as a label. It may be configured such that the solution is supplied again to the fixing portion and then a solution formed with physiological saline or the like is flowed. In this configuration, a large amount of unnecessary anucleated red blood cells can be separated and removed simply by flowing a sample through the main channel, and furthermore, rare cells that cannot be detected by particle size are separated and detected using an antigen-antibody reaction. Various diagnostics are possible.

  The physical drive means in the present invention includes, for example, physical drive, liquid filled in a syringe, drive by a pressurized pump via air, and volume change of a container in which liquid is accommodated by displacement of a piezoelectric body A pump that performs the above-described operation is exemplified, and is preferably used as a drive source by being connected to the carrier at one point.

  When the most common syringe pump is used as a means for feeding liquid to the microchannel, as described above, there is a problem that bubbles are easily mixed when the syringe and the chip are connected by a tube or the like. If the material of the chip is likely to contain air, such as polydimethylsiloxane (PDMS), the air introduced together with the specimen into the chip is absorbed by the chip by vacuuming the chip in advance. Therefore, this problem can be solved. However, such an effect cannot be expected with a hard material such as an acrylic resin, so another means is required. For example, as shown in FIG. 17, a method of providing a flow path for removing mixed air is an example. That is, the air removal channel 1703 is provided on the upstream side of the branch channel group 1702 that branches from the main channel 1701. The fluid in the main channel 1701 flows from left to right in the figure. The air removal channel 1703 communicates with the deaeration port 1704. If the resistance value of the air removal flow path 1703 is designed to be sufficiently smaller than the resistance value of the branch flow path group 1702, most of the liquid introduced into the chip will flow to the air removal flow path 1703 side. The removed air is also removed from the deaeration port 1704. If the deaeration port 1704 is closed with a stopper or the like when air removal is completed, the resistance value on the air removal channel 1703 side is infinite, that is, equivalent to a state where there is no channel, and the main channel 1701 and the branch channel group The flow advances toward 1702, and the separation process as designed is performed. In this way, the problem of air bubble mixing can be solved.

  All or part of the body fluid system solution adjustment input unit, separation / extraction unit, and analysis operation means in the present invention is formed as a recess on at least one carrier, and a member in which a liquid storage structure is formed on a sheet or a necessary portion from above is provided. Although what was joined as a lid | cover is shown, not only this but the thing laminated | stacked up and down by the through hole may be used. Examples of the material of the carrier include polydimethylsiloxane (PDMS), acrylic, and silicone.

  Furthermore, the present invention uses a laminar flow in a microchannel to input a body fluid for controlling the movement of particles, a driving unit for moving the body fluid in a laminar flow state in one direction, and driving the driving unit The flow path A having a size that does not cause the particles to branch in the moved fluid, the flow path B that branches the particles having a target size depending on the size of the particles in the fluid, and the flow paths. A biological operation system equipped with an analytical operation unit for obtaining target information from the body fluid fluid obtained in step 1, a unidirectional drive source capable of laminar flow drive, and a flow path pattern and purpose With the combined configuration with the operation region that obtains the reaction, it is possible to realize a device that can be easily handled and satisfy various diagnostic purposes.

  The first advantage of actively using laminar flow is that the detection object can be confined within a single laminar flow, so that the physical width of the flow path is greater than the flow of the detection object. It can be enlarged. For example, in one embodiment of the present invention, the laminar flow width of the sample may be less than 4 micrometers and the flow path width may be 50 micrometers. Thereby, even if it is a sample containing many particles which are easy to adsorb | suck to flow paths, such as a cell, the problem that a flow path is clogged and it becomes impossible to use can be prevented. In addition, the fact that the flow path width can be increased reduces the technical difficulty of forming the flow path, and it is easy to manufacture a stable flow path device at low cost.

A second advantage is that the sample can be concentrated by confining the sample to the laminar flow width. In fact, as will be described later, by providing means for classifying particles in a specific size range from a sample confined in a laminar flow, not only classification but also concentration and collection of particles in that size range are collected. Will surely be implemented. If it is a biological sample, it is possible to enhance the fluorescent signal for detecting them by concentrating and collecting the detection target, such as leukocytes, stem cells, cancer marker substances, etc. A reliable detection system can be realized.
The bodily fluid solution in the present invention is obtained from a living body such as sweat, blood, semen, bone marrow fluid, pleural effusion, exudate, saliva, periodontal exudate, other cell suspension, agricultural chemical-contaminated food solution, food poisoning bacteria test Solutions, tap water, drainage, rainwater, standard balls for measuring instruments, etc. shall also be included as objects to be measured.

The driving means in the present invention may be any means that provides a driving force that allows the fluid to flow in a laminar flow state at least in accordance with the cross-sectional size of the flow path. For example, a hand-held syringe, a syringe pump drive The driving force for moving the fluid in the flow path may be formed by a syringe, a micro pump for osmotic pressure, etc., and pump driving via a gas such as air or nitrogen is exemplified.
The laminar flow in the present invention may use a flow in which a plurality of flows are stacked in addition to a single flow. In this case, different types of bodily fluids are transported at the same time, and obstacles are formed on the way to branch, and the branched laminar flow is further separated and inspected by a cross-shaped branch flow path to operate on many types of particles. It may be a thing.
The flow path in the present invention is formed by branching a target particle classification and a flow path for preventing particles from being classified with respect to the main flow path. For example, a cross flow or a T-shaped cross flow Those having a road form are preferred.

That is, in the literature shown in Non-Patent Document 2 (M. Yamada, M. Seki, LabChip, 2005, 5, 1233-1239), FIG. Based on the relationship between the particle size and the distance (virtual width) w1 from the wall surface obtained by using the Hagen-Poiseuille equation in the relationship between the cross-shaped flow path shown in FIG. Use uptake into branch channel.
The virtual width w1 shown here is the distance from the wall surface in the laminar flow state, the branch channel resistance value determined from the viscosity of the liquid, the depth, width and length of the branch channel, and the branch channel It is obtained from the relationship with the main channel resistance value determined from the depth, width and length of the main channel between them, and the size of the branch channel and main channel is selectively determined according to the purpose Is done.
The present invention is based on the conditions of the main flow path and the conditions of the flow paths formed on the side surfaces of the main flow path, the flow path that does not capture particles at all, the flow path that takes in target particles, and the flow path that takes in other particles. Forming three,
When the present invention uses blood cells such as red blood cells and white blood cells as particles, the diameter of the main flow path is preferably 20 μm to 50 μm, and the flow path that is a branched flow path and does not take in particles is 10 μm to 20 μm, red blood cells The diameter of the flow path for taking in is preferably 15 μm to 40 μm or the like.

The region for acquiring information in the present invention is appropriately selected according to the obtained sample such as liquid, blood cell,
In the present invention, the information acquisition region may be a region mainly subject to examination, for example, a bioreactor configuration may be provided, and a region for performing in vitro immunity against specific cells may be set.
The present invention may use a suspension containing a target cell in addition to a body fluid.
Furthermore, the target of particles in the present invention is not limited to blood cells and platelets, and the target of particles of microorganisms, proteins, nucleic acids, and the like is not limited to blood cells and platelets, and microorganisms, proteins, nucleic acids, viruses, fungi, protozoa, bacteria As a result, the scope of application of the present invention is exemplified by Table 1, including body fluid analysis, immunoassay, diagnosis of infectious diseases, cell culture, cell separation for in vitro immunization, and the like.
In addition, the present invention enables separation of true spheres in industrial methods, that is, separation of spheres closer to true spheres having a predetermined size, which is a standard sphere used in the field of microscopy, is also possible. It can also be used to classify target standard spheres.
That is, a component adjustment input unit for adjusting and inputting a medium including a sphere,
A separation / extraction unit that separates and extracts a target sphere in the adjustment liquid that has been adjusted and input to the component adjustment input unit, and an analysis operation unit that analyzes the sphere obtained by the separation / extraction unit and outputs true sphere identification information It may be provided with a carrier provided with.
The medium is preferably a liquid inert to a sphere, and a true sphere as a standard sphere used in a measuring instrument such as an electron microscope can be separated and acquired from the sphere.

The present invention is configured by forming all or a part of the body fluid system solution adjustment input unit, the separation and extraction unit, and the analysis operation unit as a concave part on a carrier and capping the flow, such as laminar flow, direction change flow, etc. By processing, a simple but accurate operation is realized.
・ In addition, when using laminar flow, the main flow path and the branch flow path are formed in a cross or T shape, the laminar flow is formed in a fluid containing particles in the main flow path, and the flow paths of the main flow path and the branch flow path Based on the requirements of the aperture, length, fluid viscosity, etc., a flow path for branching fluid of a liquid level, a flow path for branching target particles, a processing unit for obtaining information from the obtained specimen, etc. By the combination, a diagnostic chip with a simple drive system can be obtained. By utilizing the laminar flow width on the branch flow path side for the particle selection principle, a separation threshold much smaller than the width of the main flow path or the branch flow path can be realized. Using a flow path of about μm, it becomes possible to perform size separation of particles of several μm or less.
・ In the case of the chip using hydrodynamic filtration (HDF) shown in Fig. 2 and Fig. 4, it is possible to "separate" and "concentrate" particles of a certain size. It is characterized in that it is not necessary and has little damage.

FIG. 1 shows an embodiment of the present invention. Example 1 FIG. 2 is a view showing another embodiment of the present invention. (Example 2) 3 is a cross-sectional view taken along the line X-X ′ of FIG. 2. FIG. 4 is a diagram showing another embodiment of the present invention. Example 3 FIG. 5 is a diagram showing an example of a microchannel pattern used in the present invention. (Example 4) FIG. 6 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 7 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 8 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 9 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 10 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 11 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 12 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 13 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 14 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 15 is a diagram showing another embodiment of the present invention. (Example 6) FIG. 16 is a diagram showing another embodiment of the present invention. (Example 7) FIG. 17 is a diagram showing an example of a microchannel pattern used in the present invention. FIG. 18 is a diagram showing another embodiment of the present invention. (Example 4) FIG. 19 is a diagram showing another embodiment of the present invention. (Example 4) FIG. 20 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 21 is a diagram showing another embodiment of the present invention. (Example 5) FIG. 22 is a diagram showing another embodiment of the present invention. (Example 6)

According to the present invention, the sizes of the main flow channel and the branch flow channel change depending on the information to be obtained, but the flow channel is at least large enough to form a laminar flow and the flow corresponding to the size of the particle to be obtained. A comb-shaped channel configuration that obtains a channel that does not take in the channel and particles and obtains a liquid of about the liquid component, an information acquisition unit that performs color reaction with the separated liquid, leukocytes, and immune cells such as hematopoietic stem cells Alternatively, a part of the configuration may be formed such that in vitro immunization is performed to obtain a desired immunity substance.
When leukocytes are classified, the leukocytes are counted to provide an information acquisition area (not shown in detail) on the carrier for diagnosis of various diseases such as leukemia and infectious diseases.
The laminar flow in the present invention has a high speed at the center and a slow speed as it approaches the wall surface. The width, depth, and length of the diameter of the branch channel formed on the side surface in the middle, and the mainstream in the latter stage The particle uptake width from the wall surface is determined from the relationship between the width, depth, and length of the diameter of the path, and particles having a center of gravity within the uptake width are taken into the branch channel.
By reducing the take-up width, it is possible to take in only the liquid without taking in the particles, and the principle is non-patent document 2 (M. Yamada, M. Seki, LabChip, 2005, 5, 1233-1239). (Fig. 2 etc.).

An embodiment of the present invention will be described in detail with reference to FIG.
Reference numeral 1 denotes a body fluid system solution input unit, which includes a supply unit 1a that supplies blood, saliva, cell suspension, and the like from the outside. In some cases, a body fluid system solution input unit 1 includes a diluent supply unit 1b. In some cases, mixing agitation means for mixing may be provided.
In addition, a previously adjusted dilution may be input to the body fluid system solution input unit 1.
The body fluid system solution input unit 1 can be described by replacing it with a medium input unit in the case of a medium for obtaining a true sphere.
2 is a separation and extraction unit that separates the particles on the laminar flow according to their size, separates them by electrification and magnetism, and passes or blocks the passage of particles other than those bound by the addition of labeling substances. It is.

3 is an analytical operation means, and the separation / extraction unit 2 performs operations such as the number of particles to be extracted, their properties, or a color reaction by mixing a reagent with the obtained target liquid. Then, the obtained signals, color information, etc. are output to an external processing device, processing carrier, etc. (1c).
In the case of classification of spheres, a spherical shape, a camera for judging distortion and deformation, and a non-contact shape measuring instrument using laser light are set, and the classification and measurement of leukocytes can also be used.
In addition, for a unidirectional flow in a laminar flow, it is further branched into two Y-shaped flow paths, and a path changing means using laser light, ultrasonic waves, etc. for changing the flow of particles is provided at this branch. Provide a laser beam and an ultrasonic wave so that the sign is detected before the course changing means, and the course changing means determines the branching direction of the labeled particles according to the state of the sign. The change flow obtained by making it change is also illustrated.

Reference numeral 4 denotes a driving means, which preferably outputs a laminar flow, and examples thereof include a syringe pump that is manually or automatically operated, and fluid drive by electrostriction operation using a piezoelectric body.
Although the laminar flow depends on the properties of the liquid to be moved and the shape and size of the flow channel, the laminar flow is sufficiently formed in the flow channel even when driven by a syringe pump.
Reference numeral 5 denotes a carrier, which is formed of PDMS, silicone, acrylic, or the like, and has a flow path and a processing part formed by recesses on the surface.

The configuration in the present embodiment is formed by forming each configuration in a concave shape on one carrier 5 and further laminating a lid.
In this state, the specimen or the sphere-containing medium is supplied from the supply unit 1a. After the supply, the driving means 4 is coupled to this portion and driven to form a laminar flow on the flow path.
A laminar flow can form a single layer or a plurality of layers without mixing a plurality of flows having different properties. Therefore, a plurality of processes can be performed at one time by separating and recombining each laminar flow. Different diagnosis may be possible.

If the reagents are also made into a laminar flow, it is possible to provide a configuration in which the free antibody can be supplied remotely even when the reaction tank is inside the carrier and cannot be directly supplied, or even if it is desired to have a single supply port.

The body fluid solution supplied from the supply unit 1a is supplied to the separation and extraction unit 2 to separate and extract target particles or solutions.
The extracted solution or particle is reacted with a reagent or an antibody reagent in the analytical operation means 3 and its color development state, when a fluorescent substance is added, fluorescence information, antigen-antibody reaction, particle counting, etc. The target information is obtained or provided to an external device.

In the analysis operation means 3, for example, a stimulating substance may be further added to the immunity candidate cell whose size has been detected to perform in vitro immunization and induce differentiation into antibody-producing cells.
After that, it can be taken out, DNA can be extracted, and a desired site can be detected and amplified to form and amplify a single chain antibody gene or a human type gene to produce an antibody. good.
As shown in this example, in the combination of the means for discriminating by particle size and the means for measuring specific particles by immunological technique, particles having the same particle size are distinguished. Thus, the configuration to be extracted can be shown effectively.
For example, when distinguishing between nucleated red blood cells and white blood cells, classification by classification may be difficult, but in that case, for example, the cell membrane of red blood cells bound to a labeling substance having magnetic and fluorescent properties as described above. Prepare an antibody against the antigen, leave only cells of the size of the white blood cell level in the main flow path, and provide a region that binds to the labeled antibody as the analysis operation means 3 in the subsequent stage to label only nucleated red blood cells. A method of supplementing only nucleated red blood cells with a visual or magnetic force, electric field force, antibody against the above-mentioned fixed cell membrane antigen, etc. is added, and blood cells of the same size can be identified.
When specimen preparation or the like is used, nucleated red blood cells can be visually identified by performing red blood cell staining such as Giemsa staining or Pappenheim staining, which is an example of a diagnostic chip.

A diagram specifically showing the vicinity of the separation and extraction unit 2 of the embodiment shown in FIG. 1 according to the present invention will be described in detail with reference to FIGS.
3 is a cross-sectional view taken along the line XX ′ of FIG.
Reference numeral 200 in FIG. 2 denotes a substrate, which is formed with grooves serving as micro-channels and with lids (302 shown in FIG. 2) mounted on both sides or one side.
The material is selected depending on the width of the flow path, but resin materials such as PDMS, silicone, acrylic, and polycarbonate, and glass are mainly used, but a rectangular flow path that does not require taper is formed. It is preferable in terms of simplicity of lid joining.
As the manufacturing method, for example, a method of performing a molding process by injection molding or hot embossing using a mold base formed by using a technique of photolithography or etching treatment is shown.

Reference numeral 11 denotes a sample input unit, which is a hole-like portion to which sweat, blood, semen, bone marrow fluid, pleural effusion, exudate, saliva, and the like are supplied. The sample input unit may be connected to the output unit of the driving unit after sample input.
Reference numeral 12 denotes a main flow path, which has a width of 20 μm to 100 μm, a depth of 10 μm to 50 μm, and a length of 3000 μm to 10000 μm, and differs depending on the type of body fluid, and the branch flow path 13a that intersects the main flow path 12 perpendicularly. , 13b, 14a, 14b, 15a, 15b are also formed by similar grooves, but the width, depth, and length are for classifying and taking up particles such as red blood cells and white blood cells when classification is not intended. It depends on the size of the particles.

Reference numerals 13a and 13b are liquid collection flow path groups, which are a plurality of flow paths that detect only liquid, for example, plasma without taking in particles in the specimen. These multiple flow paths are individual flow paths. Since the diameter of each is small, one processing amount is small, so that a plurality of flow paths are arranged in parallel.
The width and depth of the flow path for collecting liquid are 10 μm to 30 μm, 10 μm to 50 μm, and the length is 10000 μm to 30000 μm. The connection interval of each branch flow path is 20 μm to 50 μm.

Reference numerals 13a1 and 13a2 denote liquid storage units that temporarily store the liquid flowing from the flow path.
Reference numerals 13a2 and 13b2 denote liquid output portions, which are liquid supply portions when an immune test piece, a component color development test piece, and a transport container are inserted.
The part is often very small and may require a device for sucking from the outside and a capillary suction device.
Reference numerals 14a and 14b denote red blood cell separation flow path groups, which are connected to the main flow path 12 in the same manner, although the width, depth, and length are different from the liquid collection flow path. The width of the red blood cell separation channel is 15 μm to 50 μm, the depth is 10 μm to 50 μm, and the connection interval of each branch channel is 20 μm to 50 μm.

14a1 and 14b1 are red blood cell reservoirs for temporarily storing red blood cells, and 14a2 and 14b2 are vent holes and also are extraction holes for taking out red blood cells to the outside.
Since the number of red blood cells is very large, a large number of channels are formed.
Reference numerals 15a and 15b denote white blood cell collection flow path groups, in which white blood cell collection flow path groups 15a and 15b having a width, a depth, and a length for separating and collecting white blood cells are formed.
The width and depth of the leukocyte collection channel are 20 μm to 50 μm, the length is 10000 μm to 20000 μm, and the connection interval of each branch channel is 20 μm to 50 μm. The leukocyte collection channel group in the figure is the leukocyte collection channel group 15a and the lower leukocyte collection channel group 15b, and the number, width, and depth of the channels are different and differ depending on the type of leukocytes. It has a structure that can sort the size.

１５ａ１、１５ｂ１は、白血球貯留部であり、一時的に集められ濃縮貯留される部分である。１５ａ２及び１５ｂ２は、通気口であり、場合によっては、白血球取り出し部にもなる。
１６は、白血球操作部であり、得られた白血球を濃縮し、その容積から計数したり、形状、色等で白血球の種類を更に細分化したりする領域を示したものである。
又、予め標識を付けた特定の抗体と抗原の結合した遊離抗原を供給し、この遊離抗原と抽出した白血球との結合の有無による免疫学的手法により、白血球の種類や、各種の免疫疾患の診断も可能である。
１７は、残余粒子検出部であり、造血幹細胞等の特殊な細胞などを検出するための部位である。
１８は、免疫試験片Ａであり、図３で示す試験片挿入部Ａ３０４に免疫試験片を挿入する。 Leukocytes may be accompanied by manipulations for collecting antibody genes by in vitro immunity.
If the size varies depending on the type of leukocyte (neutrophil, eosinophil, basophil, monocyte, lymphocyte, B cell, T cell, NK cell, etc.) (for example, particles other than leukocytes) 1) and 15a and 15b in FIG. 1, the arrangement may be such that the width, depth and number of collection channels of the leukocyte collection channel group are changed.

15a1 and 15b1 are leukocyte reservoirs, which are temporarily collected and concentrated and stored. 15a2 and 15b2 are vent holes, and in some cases, they also serve as leukocyte extraction units.
Reference numeral 16 denotes a leukocyte operation unit, which shows an area where the obtained leukocytes are concentrated and counted from the volume, or the types of leukocytes are further subdivided by shape, color and the like.
In addition, a free antigen in which a specific antibody labeled with an antigen and an antigen are bound is supplied, and an immunological technique based on the presence or absence of binding between the free antigen and the extracted leukocyte, the type of leukocytes and various immune diseases Diagnosis is also possible.
Reference numeral 17 denotes a residual particle detection unit, which is a part for detecting special cells such as hematopoietic stem cells.
Reference numeral 18 denotes an immune test strip A, which is inserted into the test strip insertion portion A304 shown in FIG.

An immunological test strip is a test paper whose color changes when an antibody adheres to a so-called test paper and binds to an antigen, and examples thereof include a commercially available immunochromatographic test strip.
Reference numeral 19 denotes an immune test strip B, which is an introduction portion of a test strip for immunoassay different from the immune test strip A18 as necessary. As shown in FIG. 3, it is used in a state of being inserted into the test piece insertion portion B305.
Immunochromatography refers to immunochromatography, which is commercially available from Fujirebio Inc., and uses capillary action to move the sample, pass it through the test area containing the free antibody bound to the label, and then immobilize it. In the antibody region thus formed, the antigen to which the labeled antibody is bound is bound at the fixed antibody portion to form a visible line by the label.
If there is no target antigen, a line cannot be formed, and if there is a target antigen, a label color line is formed. This can range from a visible level to an optical sensor, and an optical sensor can be used when accuracy is required.
In the case of using an ELISA kit or the like, a container for transporting the obtained plasma may be substituted.
Furthermore, a region including the labeled free antibody is set in advance directly in the middle of the flow path without using the liquid movement utilizing the capillary force of a porous sheet such as a conventional nonwoven fabric, and then The antibody region fixed in the reservoir may be set,
Furthermore, there may be a case where a similar free antibody region and a fixed antibody region may be set for the flow path for taking in particles. Furthermore, the same antibody may be arranged also in the main channel.

  In some cases, the immunological test strip A18 and the immunological test strip B19 may be connected to each other by a flow path formed on the bottom surface or around the edge of the carrier to compensate for the liquid that is insufficient in the separation of one side. Reference numeral 301 denotes a driving unit which is attached to the sample input unit 11 before and after the sample is supplied, and forms a laminar flow of the liquid containing the sample in the main channel 12. Reference numeral 303 denotes a support unit that supports and fixes the substrate 200, and the body fluid system solution input unit 1 and the analysis operation means 3 in the previous stage are also supported and fixed on the same support unit 303. The size of the substrate is approximately 200 mm @ 2 to 2000 mm @ 2. The determination of the above numerical values is within a selection range as appropriate.

Next, the operation of the separation / extraction unit shown in FIG. 2 will be described.
A sample made of blood or diluted blood is supplied from the sample input unit 1, and an external driving unit 301 shown in FIG. 3 is connected. The external drive unit 301 pushes blood from the sample input unit 11 toward the main flow path 12 by pushing physiological saline with a syringe pump.
The blood or diluted blood in the main flow path flows from left to right in the figure in a laminar flow state, but the liquid branches in the liquid collection flow path groups 13a and 13b, and the liquid in the liquid collection flow path group 13a The liquid is stored temporarily in the liquid storage part 13a1, moves to the mounted immunological test strip B19 via the liquid output part 13a2, and the liquid in the liquid collection channel group 13b is temporarily stored in the liquid storage part 13b1. Then, it moves to the immunological test strip A18 inserted into the test strip insertion section A304 and the immunological test strip B19 inserted into the test strip insertion section B305 via the liquid output section 13b2, and reaches the respective immunological test strips to supply the impregnation. Is done. Each immunological test strip is made of a porous material and passes through the antibody line while impregnating the inside. In this case, when the antigen in the liquid matches with the specific antibody line, it binds there. In this part, light and shade occurs in that part, and it is a mechanism that can understand what kind of antigen is contained.

On the other hand, the test piece may be used for a glucose test impregnated with this liquid and used in a dry chemistry or a wet chemistry that develops color by reacting with a reagent that reacts with a blood component that has been preliminarily disposed.
The blood in the main flow path 12 that has passed through the liquid collection flow path groups 13a and 13b enters and moves into the flow path groups of the red blood cell separation flow path groups 14a and 14b, and is classified and separated.
Red blood cells that have passed through the red blood cell separation channel groups 14a and 14b are temporarily stored in the storage units 14a1 and 14b1.
The blood that has passed through the region of the red blood cell separation channel group 14a, 14b is separated into white blood cells in the region of the white blood cell collection channel group 15a, 15b by laminar flow, and temporarily stored in the white blood cell reservoirs 15a1, 15b1, respectively. . The temporarily stored white blood cells move to the white blood cell operation unit 16.
The leukocyte reservoir 15a1 may be the leukocyte operation unit 16 as it is.

In the leukocyte operation unit 16, the number of leukocytes to be detected is different between the upper and lower sides, and therefore the number of the white blood cells and the size of one channel are different.
In some cases, a counting counter sensor may be connected in the middle of the leukocyte operation unit 16.
The finally remaining particles and the like are collected in the residual particle detector 17. In this part, there may be a case where a larger cell may exist, so that it can be collected and subjected to a secondary inspection.
In this embodiment, a chip-like substrate 200 made of, for example, PDMS is formed as a recess, and finally a glass sheet is bonded as a lid 302 except for an input portion, a take-out portion, and the like.

Furthermore, another embodiment of the separation and extraction unit shown in FIG. 2 of the present invention will be described with reference to FIG.
2 to 4 can be an embodiment of the present invention depending on circumstances.
Reference numeral 41 denotes a sample input unit, which has a configuration similar to that shown in FIG. Reference numeral 42 denotes a buffer introduction part for forming different laminar flows so that blood in the main channel 43 flows more in a state closer to the branch channel.
Reference numeral 44 denotes a branch channel for liquid extraction. A plurality of channels having a depth and a width that do not allow particles to enter are formed in parallel and collected by the liquid extraction unit 45.
As in FIG. 2, the liquid extraction unit 45 has a supply port for supplying a liquid component to the test piece 45P mounted in the downward direction.
Reference numeral 46 denotes a branch channel for separating red blood cells, which mainly has a depth and a width for taking red blood cells into the branch channel 46 for separating red blood cells.
47 is a part for temporarily accumulating red blood cells extracted in the branch flow path. 47a is a vent.

Reference numeral 48 denotes a leukocyte separation branch channel, and a plurality of channels having a width and a depth for capturing leukocytes in the branch channel are formed. When the size of leukocytes changes depending on the type, a flow path with a different diameter may be connected.
Reference numeral 49 denotes a white blood cell counting unit, which is formed by arranging a sensor for optically, magnetically, and electromagnetically counting the number of flowing white blood cells.
Reference numeral 50 denotes a leukocyte separation unit, which may be counted from the volume of leukocytes separated and concentrated in this part. 50a is a vent.
In addition, blood diseases can be diagnosed by measuring the shape, size, color, and distortion of white blood cells using optical means.
51 is a specific cell detection unit, a part where particles that have not been detected in the flow path are stored,
For example, hematopoietic stem cells may accumulate.
These structures are also formed on one substrate 400 as in the embodiment shown in FIG.

The embodiment shown in FIG. 4 uses only one of the bidirectional flow paths of FIG. 2. Therefore, a buffer is introduced from the buffer introduction section 42 to form two laminar flows, and blood in the main flow path 43 is obtained. Is for the branch channel side. By introducing this buffer, the efficiency of capturing liquid and blood cells is improved. The supply amount of the buffer buffer is appropriately selected in the range of 1: 1 to 1: 5, compared with the sample amount.

In the main channel 43, blood or diluted blood flows in a laminar flow state on the side of the branch channel. In the liquid extraction branch channel 44, the red blood cell separation branch channel 46, and the leukocyte separation branch channel 48, respectively. Each fluid and particle is supplemented by hydrodynamic action.
The embodiment shown in FIG. 4 can perform the above-described function without requiring a buffer, and may be unnecessary when detecting mainly a plasma component.
In addition, the portion where blood or liquid other than particles in diluted blood, mainly blood plasma, is extracted does not necessarily have to come first, the latter stage of the red blood cell extraction branch group, the white blood extraction branch flow In some cases, it may be a later stage of the road.

Other configurations

The present invention falls within the category of lab-on-the-disk (chip), and is not limited to body fluids. For example, organs, blood, skin, and the like that contain many immune cells such as spleen cells, bone marrow cells, hematopoietic stem cells, and peripheral blood lymphocytes. Cell suspension such as a solution containing cells excised from the living tissue.
In addition, the reason why the body fluid system is used is that it may be effectively used in plant units even in plants.
That is, the present invention provides not only the analysis but also the immunization preparation process in which extracorporeal immunization is performed on B cells, lymphocytes, etc., which are white blood cells such as antibody-producing cells. Alternatively, it may be formed before or after the analysis operation means 4.
This point will be described in detail.
For example, an organ cell suspension is input to the sample input unit 11 of the separation / extraction unit shown in FIG. 2 to separate red blood cells in the branch flow path group, and white blood cells such as B cells are collected into white blood cell collection flow path groups 15a, 15b, It may be obtained by the residual particle detector 17, and here, a liquid such as a remaining plasma component may be collected.

A region for performing in vitro immunity may be formed by adding cytokines, stimulating substances, and antigens to the B cells and lymphocytes at the leukocyte operation unit 16.
After the addition, the cells are cultured for several days to several weeks in a thermostatic chamber, so that an incubator or an equivalent thereof may be required separately.
The additive forms a reservoir and can be added automatically. Examples of stimulating substances include IL-2, IL-4, IL-5, IL-10, MDP, anti-CD38, muramyl dipeptide, anti-CD40,
Examples of antigens include tumor antigens, bacterial cell membrane components, rice allergen, casein, hoboalbumin, h (human) S100 family, he (chicken) EL, h (human) Ras, mite extract, h (human) rap74, h Examples include (human) TOPO2B, cedar pollen, proteins other than mice such as b (bovine) SA, b (bovine) Casein, and proteins such as mSA and mMapk1.
After supplying these additives, these immune cells are induced to differentiate into memory B cells or antibody-producing cells depending on their properties, and affinity maturation class switching of antibodies is highly practical. It becomes a cell from which immunoglobulin can be obtained.
In some cases, white blood cells obtained from peripheral blood such as peripheral blood human lymphocytes may be used.

Furthermore, the antibody can be produced in a short time by combining the step of obtaining an antibody by colonizing it with a host cell such as Escherichia coli. These genetic manipulations are difficult on the chip, but when producing antibodies in host cells, a small bioreactor function and incubator function are combined to form a lab-on-the-disk (chip). Is also possible.
The cells are selectively removed, and antibody genes (VH gene, VL gene, etc.) obtained from the cells are amplified by PCR (polymerase chain reaction) method to form a single chain antibody (scfv) or the like.
These antibody production methods are described in JP 2006-180708 A, JP 2004-121237 A, Chemistry and Biology, (2007, November), vol. 45no. 11, p751-753, Abstract of BMB2008 Lecture, published on November 20, 2008, 1P-1344, and other descriptions can be used in the present invention as well.

Furthermore, another embodiment of the separation and extraction unit shown in FIG. 2 of the present invention will be described with reference to FIG.
The purpose of this example is not to collect particles in a specimen but to collect only liquid components that do not contain particles of a certain size or larger. For example, only plasma components from blood are collected. Applicable when collecting. Common methods for recovering only plasma components include centrifugation and a filtration filter that uses a mesh structure, etc., but the centrifugal method is large and integrated with the plasma component detection system. In addition, the filtration filter limits the amount of processing because clogging of particles such as blood cells occurs. If only the plasma component can be recovered on the chip as in the present invention, it is easy to integrate a detection unit that uses a reagent or fluorescent light emission with respect to the plasma component on the recovery unit or its extension. In addition to the miniaturization, it is possible to complete the processing without touching the specimen at all by the examiner, so that there is an advantage that the safety is high. In addition, as described above, since a flow channel structure having a width larger than the size of particles to be separated can be used by using laminar flow, in principle, clogging does not occur, and the processing amount is limited. It also has the advantage of not.

FIG. 5 shows a specimen introduction part 501 on a carrier 50T made of a flat plate made of PDMS, acrylic or the like.
When the main flow path 502, the branch flow path group 503, the vent 504, and the plasma collection part 505 are respectively formed by forming cylindrical and rectangular parallelepiped recesses, and the carrier 50T is formed of, for example, PDMS A tubular channel or the like is formed by bringing a surface on which a recess is formed on a flat glass plate into contact with each other.
Specifically, first, the sample is introduced from the sample introduction unit 501. As in Example 3, for the purpose of separating and recovering the particles, a structure in which the particles are pressed with a buffer flow so that the particles can easily enter the laminar flow on the branch flow path side is required. If the purpose is not to collect, the buffer flow may not be present and the buffer introduction can be omitted. The introduced specimen such as blood travels through the main flow path 502 and reaches the vent 504, but only the liquid component not containing particles such as blood cells is collected in the branch flow path group 503 along the way. The flow path width of the branch flow path group may be larger than the size of the particles to be excluded, and the laminar flow width is such that the particles to be excluded do not enter the laminar flow entering the branch flow path side with respect to the resistance value of the main flow path. It suffices if the branch channel has a resistance value for forming the. The liquid component that does not contain particles separated in this way is collected from the plasma collection unit 505.

  When collecting plasma components from blood samples, the plasma components are designed so that particles with a diameter of 1 μm or more do not enter the branch flow path side so that particle components having a size larger than red blood cells are not mixed in the plasma components. Do. 5 differs from FIG. 2 in that a branch channel group 503 is provided only in one direction with respect to the main channel 502. This means that the structure should be properly used depending on the amount of liquid to be flowed to the branch side (that is, the liquid to be collected). If the flow rate on the branch side is to be increased, the number of branch flow paths can be increased, or as shown in FIG. In this way, the branch channels may be provided in a plurality of directions with respect to the main channel. This point is common in the following embodiments.

  In addition, even when only liquid components such as plasma are desired to be collected, there are cases where impurities having a size larger than that of the main flow path are present when handling a sample having a large amount of impurities such as that obtained from nature. sell. When the main flow path is clogged with such impurities, the resistance value balance changes, and a phenomenon occurs in which particles that would otherwise not enter the branch flow path are recovered. In order to prevent the occurrence of such a problem, it is preferable to form a filter structure having a larger gap than the main channel between the sample introduction unit 501 and the main channel 502 so that impurities can be removed in advance. . Alternatively, by using a laminar flow separation, it is possible to form a filter structure using the physical void size in the downstream area of the chip for the collected sample having a certain size, and perform the second-stage separation. Good. Even if there is a risk of clogging in a filter using physical voids due to the presence of many particles in the initial stage, there will be no large size particles after separation using laminar flow. This is because the number itself is also decreasing, and there are many cases where the risk of clogging has disappeared.

  Unlike the example of FIG. 5, when it is desired to positively collect particles such as cells in the specimen from the branch channel, a channel configuration as shown in FIG. 18A is useful. Unlike the example of FIG. 2 or 5, in this case, in addition to the sample introduction unit 1801, a buffer introduction unit 1802 is connected to the main channel 1803 upstream of the branch channel group 1804. A sample containing particles to be separated and recovered is introduced from the sample introduction unit 1801, while a buffer solution not containing particles is introduced from the buffer introduction unit 1802. In the case of such a configuration, as shown in FIG. 18B, the liquid from each introduction portion contacts at these connection points to form an interface 1808. Since the particles contained in the specimen are pressed against the branch channel group 1804 side by the buffer flow, the separation efficiency is improved. This is because the fact that particles that cannot be included in the laminar flow width on the branch flow path side cannot enter the branch flow path is an event that occurs regardless of the existence of the buffer flow, but particles that are included in the laminar flow width on the branch flow path side. This is because whether or not actually enters the laminar flow is determined by whether or not the particles are present near the wall surface on the branch flow path side. That is, the separation efficiency can be increased by preliminarily pressing the particles near the wall surface on the branch flow path side by the buffer flow. Also in the following embodiments, when it is basically desired to separate and collect particles from the branch flow path, it may be considered that there is a buffer introduction part in addition to the specimen introduction part. In the system of the present invention, since the laminar flow width on the branch channel side is controlled by the channel resistance value, it is easy to provide a plurality of branch channel groups having different separation size ranges on the same chip. Particles in one stage separation size range are collected from the first separation / recovery unit 1806, particles in the second stage separation size range are collected from the second separation / recovery unit 1807, and so on. In addition, large particles that do not fall within any separation size range travel straight through the main flow path 1803 and are collected from the non-branch collection unit 1805.

  Thus, whether particles having a certain size enter the laminar flow on the branch flow path side is separated not only by the width of the laminar flow but also greatly influenced by the position of the particles. A corresponding laminar width design is required. For example, it is assumed that a relationship of a <b is established in a specimen including a particle group having an average radius (short diameter) a and a particle group having an average radius (short diameter) b. If you want to separate and collect particles with an average radius (short diameter) a from the branch channel, logically design the channel pattern so that the laminar flow width on the branch channel side is a. Since only the particles of a can enter, the particles can be recovered. However, with such a separation threshold, particles whose radius is slightly larger than the average value a, or particles whose location is slightly separated from the wall surface on the branch channel side, even if they are the same type of particles, Since it cannot enter the stream, the separation and recovery efficiency is significantly reduced. In order to increase the separation and recovery efficiency, it is necessary to provide a certain margin for the laminar flow width on the branch flow path side. For example, in the separation of a specimen containing two particle groups having an average radius of a <b, the laminar flow width on the branch channel side is a + (ba) × k (k is an adjusted value). Only the particle group having the average radius a is separated to the branch channel side by the branch channel. If the adjustment value k is set to a value between 0.2 and 0.8, the target separation can be realized with high efficiency. In general, other particle groups are also included, but if the average radius a of the particle group that is desired to be separated and recovered from the branch flow path is b, In addition, since the above relationship is established, the same design may be performed for the flow path pattern that performs separation in multiple stages. FIG. 18 (a) is an example in which two-stage separation is performed. The average radius of the particle group contained in the specimen is a, b, c (a <b <c, where c is a plurality of types of particles larger than b. Group, and a may be a plurality of particle groups smaller than b), the first stage separation / recovery unit 1806, the second stage separation / recovery unit 1807, and the non-branch collection unit 1805 What is necessary is just to design so that the particle group of a, b, and c may advance to each. That is, the laminar flow width m in the branch channel group leading to the first stage separation / recovery unit 1806 is designed so that a <m <b, and the layer in the branch channel group leading to the second stage separation / recovery unit 1807 is designed. The flow width n may be designed so that b <n <c.

  Here, an example of setting the separation size range of the branch channel group according to each sample and the purpose of separation will be described. When the most common blood for clinical use is a specimen, the separation size range of the first-stage branch channel group located at the most upstream is (3-α) μm or less (where α satisfies 0 ≦ α ≦ 2) In this case, only plasma components that do not contain blood cells can be collected through the branch channel group. This means that the width of the laminar flow on the branch flow path side formed in the main flow path is designed to be (3-α) / 2 at the position of the branch flow path group. Similarly, when the blood is a specimen, the separation size range of the branch channel group is set to (7−α) μm or less (where α is any value satisfying 0 ≦ α ≦ 2), so that blood cell components Among them, only red blood cells can be collected through the branch channel group. Further, a branch whose separation size range is set to (10 + α) μm or less (where α is any value satisfying 0 ≦ α ≦ 4) on the downstream side of the main flow channel from such a branch flow channel group for red blood cells. By arranging the flow path group, a particle group mainly composed of white blood cells not containing red blood cells among blood cell components can be collected through the branch flow path group. In addition, the separation size range of the first-stage branch channel group is set to (10 + α) μm or less (where α is any value satisfying 0 ≦ α ≦ 4), and is downstream of the main channel from the branch channel group. The separation size range of the second-stage branch channel group arranged on the side is set to (20 + α) μm or less (where α is any value satisfying 0 ≦ α ≦ 5), so that it is more average than white blood cells. Particles such as large stem cells, circulating tumor cells, iPS cells, and various cell masses can be collected through the branch channel group. Further, by having a flow path pattern in which m types of branch flow path groups having different size separation ranges by n are arranged, an analysis chip that requires a resolution n and a particle size distribution with a step number m can be realized.

  As described above, the configuration of the flow path as shown in FIGS. 2, 4, 5, and 18 is a typical example of the present invention. When only such a physical configuration is used, the laminar flow width is controlled. Particle size separation by doing this is a basic function. In practice, however, there are many types of particles contained in the specimen, and even if the types are the same, the particle size usually has a certain distribution. Therefore, there are many cases where sufficient separation cannot be obtained even if the particles are separated only in the dimension of size. Actually, the physical or chemical or biological substance different from the size in the part where the particle group after the separation based on the size is passed or stored, such as 14b1 in FIG. 2 or 47 in FIG. Based on the physical properties, a function of further separating the particle group is required.

  In particular, when it is desired to collect particles having a very low frequency in the specimen, such a function is very effective. For example, if you want to recover particles that are present in the sample only in an amount of about 0.01% or less from the group of particles contained in the sample, even if you try to recover the original sample using an antibody, Therefore, there is a high possibility that the antigen-antibody reaction is not appropriately performed. Even when a method capable of detecting individual particles such as flow cytometry is used, there are too many particles other than the target, so that detecting individual particles is too time-efficient. When aiming at particles with a low presence frequency, in order to prevent a situation in which none of the particles are collected, the amount of the sample itself must be increased, and the inefficiency becomes more conspicuous. Even when a filter using a mesh structure is used, it is not easy to increase the amount of the specimen because clogging is caused, and the target particles may be buried in other particles and cannot be detected.

  For this purpose, the system of the present invention functions very effectively. By first performing size separation, most of the particles having a size different from the target particles can be eliminated. As a result, even if the particles are present in only about 0.1% or less of the total particles contained in the original specimen, they are present in about 1% or more with respect to all the particles in the separated liquid. Concentration can be performed to the extent. If the presence frequency can be increased to this extent, it becomes easy to use a general technique such as detection using an antibody. For example, in the sample storage part (or passage part) after size separation as shown in 47 of FIG. 4, it is possible to store an antibody supplementing the target particle and store the particle in this part. . Alternatively, an antibody that supplements particles other than the target particles may be arranged in this portion, and only the target particles may advance to the outlet (vent hole 47a). Further, a narrow flow path may be provided between the storage unit and the outlet, and individual particles may be detected in this flow path as in flow cytometry. This is because the presence frequency of the target particles is high here, and the efficiency is high even in the method of detecting individual particles. Labeling with a fluorescent label or magnetic beads necessary for detection may be performed in the storage unit.

An example of size separation and separation or collection with an antibody will be described in more detail with reference to FIG. When the trap region 1903 is disposed between the separation / collection unit 1901 and the branch flow channel group 1902 leading to the separation / recovery unit 1901, unnecessary particles out of the size range particles entering the branch flow channel group 1902 are removed. The trap region 1903 may be supplemented so that only particles to be collected can pass to the separation and collection unit 1901. With this configuration, it is easy to collect the particles, but care must be taken because there is a possibility that the trap region may be clogged if there are too many unnecessary particles. Since the branch flow channel group 1902 is often designed to be thin for the purpose of adjusting the resistance value, the trap region is not located in the branch flow channel, but is located upstream of the separation and recovery unit in these downstream regions. If the assumed particle is a cell, the trap region 1903 may be provided with an antibody that supplements only unnecessary cells. On the other hand, when the trap region 1905 is disposed in the separation / recovery unit 1904, either a configuration in which unnecessary particles are supplemented in the trap region or a necessary particle in the trap region is supplemented is possible. In the former case, if the recovered material is sucked from the separation / recovery unit 1905, it is possible to recover mainly the necessary particles, but there is a possibility that unnecessary particles that could not be captured may also be mixed. In the latter case, the necessary particles remain in the separation / recovery unit 1905 after the collected material is sucked, so that it is necessary to separately collect the particles. Note that the substance that binds to the necessary particles in the separation and recovery unit 1905 does not necessarily have to be fixed. For example, when an antibody linked to magnetic particles or fluorescent particles is bound to necessary cells, the necessary cells may be collected using a magnetic field or may be selectively collected using fluorescent light emission. When performing such labeling on necessary particles, only particles with specific fluorescence downstream of the trapping region are distributed to another flow path with laser light, or only particles labeled with a magnetic field are specified. You may combine with the structure which advances to this flow path.

  As an application example of the collection of particles with low presence frequency, there is a collection of fetal nucleated red blood cells contained in maternal peripheral blood. Most of the maternal red blood cells are anucleated red blood cells, but when pregnant, the nucleated red blood cells made on the fetus side are slightly mixed into the mother through the placenta. If this can be collected and used for diagnosis, diagnosis can be made with peripheral blood without collecting amniotic fluid from the mother's body, and thus a very simple and highly safe diagnostic method can be realized. However, fetal nucleated red blood cells contained in maternal peripheral blood are very few, about 1-2 per ml, and considering that there are about 10 9 power per ml of anucleated red blood cells, It is very difficult to recover nucleated red blood cells. However, since nucleated erythrocytes are larger in size than nucleated erythrocytes by the amount of nuclei, size separation is first performed using this to obtain a sample that does not contain nucleated erythrocytes. Specifically, the nucleated red blood cells have a diameter of about 10 μm while the non-nucleated red blood cells have a diameter of about 7 μm. Therefore, in the flow path used in the system of the present invention, the size separation is first performed. It has a function to be called. Since erythrocytes are closer to a disc shape than a spherical shape, they can actually enter even if the laminar flow width on the branch flow path side is about 2 to 3 μm. Do the design. That is, if the main channel width is W, a fluid component having a width of 2 to 3 μm enters the branch channel side from the wall surface on the branch channel side, and the remaining fluid component having a width of W− (2 to 3 μm) is present. The ratio of the resistance value on the main channel side and the resistance value on the branch channel side is designed so that the main channel goes straight as it is. Nucleated erythrocytes are thicker than they have nuclei, and cannot enter the laminar flow width on the branch side. 20 to 100 branch channels are provided in such a region having a laminar flow width on the branch channel side so that the non-nucleated red blood cells are reliably distributed to the branch channel side. The reason why such a large number of branch flow paths is prepared is that there are a large number of non-nucleated red blood cells, and there is a high possibility that some of them will interfere with each other at the branch point and cannot enter the branch flow path.

After separating the non-nucleated red blood cells in this manner, the laminar flow width on the branch flow path side is further designed to be any value in the range of 3 to 5 μm on the downstream side of the main flow path. This is designed so that particles of any size in the range of 6 to 10 μm enter the branch flow path after removing the non-nucleated red blood cells. Nucleated erythrocytes are distributed to the branch flow path in this range, but because the size distribution of white blood cells overlaps, the sample obtained by such size separation contains a small number of nucleated red blood cells in many white blood cells. It will be a thing. The smaller the laminar flow width on the branch flow path side in the second stage, the less the number of white blood cells. However, the probability of nucleated red blood cells going straight without entering the branch side increases, so be careful when setting the laminar flow width. Cost. In any case, since the sample obtained by the second-stage separation contains leukocyte nucleic acid derived from the maternal body, fetal chromosome diagnosis or genetic diagnosis cannot be performed as it is. Therefore, for samples that have entered the reservoir by separation in the second stage, only nucleated red blood cells are collected using antibodies, white blood cells are captured and only nucleated red blood cells are passed, or dielectrophoresis Therefore, it is necessary to separately have a function of guiding nucleated red blood cells to a specific outlet. However, since the anucleated red blood cells have already been removed in the first stage, the antibody used for the second stage separation does not necessarily need to be specific for nucleated red blood cells, and the reaction differs between red blood cells and white blood cells. You can use something. In this case, generally used antibodies against protein antigens such as Glycophorin A, Band 3, Anion Exchanger 1, common leukocyte antigen, epsilon-globlin, transferrin receptor, integrin-associated protein, thrombospondin receptor, complement receptor 1 are used. be able to.
Even in the dielectrophoresis method, the charged state is different between red blood cells and white blood cells. Therefore, any sample after removing non-nucleated red blood cells can be used for the purpose of collecting nucleated red blood cells.

  The same effect can also be expected when aiming at particles whose significance is that they can be detected while the presence frequency is low. For example, it is a cell that can be used as a cancer marker, and the case of detecting circulating tumor cells (CTC) can be mentioned. In addition to having a large size distribution of 10 to 20 μm for red blood cells and white blood cells, CTC can be an indicator of cancer metastasis even if the frequency is about 2 in 7.5 ml of blood sample. A detection method using an antibody or the like is an extremely effective example. When CTC detection is performed in the system of the present invention, the size separation at the first stage is set to be performed at any value in the range of 8 to 10 μm. That is, the resistance value ratio between the branch flow path and the main flow path is designed so that the first-stage branch side laminar flow width is 4 to 5 μm. Since there are too many unnecessary red blood cells, it is necessary to provide about 30 to 100 branch channels in this range. The size separation at the second stage is set to be performed at any value in the range of 16 to 22 μm. That is, the resistance value ratio between the branch flow path and the main flow path is designed so that the laminar flow width on the branch flow path side in the second stage is 8 to 11 μm. As a result, the sample obtained from the second-stage branch flow path is a mixture of many white blood cells and several CTCs. On the other hand, a function of supplementing or passing only the CTC using an antibody or the like is also provided in a portion corresponding to the storage unit 47 in FIG.

When the number of collected cells is very small, such as nucleated red blood cells and CTCs, it is often necessary to enhance the signal using the polymerase chain reaction (PCR) method after the collection. Therefore, it is preferable that the microchannel chip used for these purposes has a structure capable of destroying cells at a site where the cells are collected and amplifying the obtained nucleic acid on the chip by the PCR method. Alternatively, the structure may be such that the signal is enhanced by performing cell culture on the chip without using the PCR method. In the case of having a function of performing cell culture on a chip, when stem cells, iPS cells, and other undifferentiated cells are cultured, properties such as the potential state of the cell surface often change after the differentiation. Therefore, there may be further provided means for separating cells by using dielectrophoresis, electrophoresis, antigen-antibody reaction, etc., after the cell culture site.

As described above, the present invention uses a channel chip that exhibits a high effect for the purpose of detecting particles having a very low presence frequency in a specimen. Specifically, it is as shown by 1 to 16.
1. 1. It has a function of eliminating particles having a size smaller than the target particle by the first-stage branching channel group disposed on the side of the main channel upstream of the main channel. It has a function of separating particles in a size range including the target particles by a branch channel group in the second and subsequent stages disposed downstream of the main channel in the first stage branch channel group.
3. The size separation function of the branch channel group controls the width of the liquid flow that proceeds to the branch channel side.
4). The separation size range of the first-stage branch channel group is set to (3-α) μm or less (where α is any value satisfying 0 ≦ α ≦ 2), so that the specimen is blood. Only plasma components that do not contain blood cells can be collected through the branch channel group.
5. The separation size range of the first-stage branch channel group is set to (7−α) μm or less (where α is any value satisfying 0 ≦ α ≦ 2), so that the specimen is blood. The red blood cells in the sample are excluded through the branch channel group.
6). Red blood cells are excluded by the n-th branch channel group, and the separation size range of the (n + 1) -th branch channel group arranged downstream of the n-th branch channel group with respect to the main channel is By setting (10 + α) μm or less (where α is any value satisfying 0 ≦ α ≦ 4), the particle group mainly composed of white blood cells not containing red blood cells becomes the (n + 1) -th branch channel group. Is eliminated through.
7). The separation size range of the n-th branch channel group is set to (10 + α) μm or less (where α is any value that satisfies 0 ≦ α ≦ 4), and is located downstream of the branch channel group from the main channel. By setting the separation size range of the arranged (n + 1) -th branch channel group to (20 + α) μm or less (where α is any value satisfying 0 ≦ α ≦ 5), the average size is higher than that of white blood cells. Particles such as large stem cells, circulating tumor cells, iPS cells, and various cell masses can be collected through the branch channel group.

8). For a liquid sample containing a plurality of types of particle groups, a means for separating the particle groups according to the difference in particle size, and a fraction containing the particle groups limited to the same size range obtained through the means Among the components, it has a function of supplementing target particles using properties other than size, or supplementing other than target particles.
9. The means for separating the particle group according to the difference in the particle size is the organism realized by advancing a part of the liquid sample traveling in the main channel to the branch channel connected to the main channel. Operation system and industrial operation system.
10. The means for capturing the particles is by using an antigen-antibody reaction.
11. The means for capturing the particles is by dielectrophoresis or electrophoresis.
12 The ratio of the target particles to be present is 0.1% or less of the total particles contained in the specimen.
13. The target particles are nucleated red blood cells in blood.
14 The target particles are circulating tumor cells in the blood.
15. For cells collected at a specific site on the chip by size separation, there are means for extracting nucleic acids in the cells and a function for amplifying the nucleic acids using the polymerase chain reaction.
16. It has a function of allowing cells collected at a specific site on the chip by size separation to be cultured on the chip.

Until now, the description has focused on the use target of the system of the present invention, but in practice, the structure of the microchannel chip used in the system itself, the structure for introducing the sample into the microchannel, Various devices are also required for the method of detecting the separated sample. Here, the structure of the microchannel chip and the method for introducing the specimen into the chip will be described in detail with reference to the drawings.
In general, since many microchannel structures are still in the research stage, a tube is attached to a flat device as shown in Fig. 3 by bonding or the like, and this is sent while controlling the flow rate like a syringe pump. In many cases, an experiment is performed by connecting a device capable of liquid. However, bubbles tend to get mixed in when connecting tubes, and when bubbles enter a micrometer-level structure, the surface resistance is large and the tube stays in the flow path for a long time, so it is often impossible to obtain the performance as designed. Occurs. At the research level, by getting used to the operation, it becomes possible to connect the tube so that almost no air bubbles are mixed in. However, if anyone uses it, the same results can be obtained without failure. A device for that is, for example, a chip structure as shown in FIG. A concave micro-channel pattern 602 is formed on the channel carrier 601 such as PDMS and acrylic, and the surface of these materials is used as necessary for bonding with glass, acrylic, polycarbonate, polypropylene, cycloolefin polymer, and PDMS. It is the same as in FIG. 3 that the flow path portion is formed by joining a plate 603 formed by applying a silica film to the substrate.

  Further, in the structure of FIG. 6, a cylindrical liquid reservoir 604 formed of a plastic material such as acrylic is formed around the entrance / exit of the flow path. This may be joined to the channel carrier 601 or may be formed by integral molding. As already described, there may be a plurality of inlets or outlets in the microchannel used in the system of the present invention, but the concept is the same as in FIG. When supplying this chip to the user, the flow path is filled with a buffer suitable for the purpose, and the liquid level 605 of the buffer is positioned below the liquid reservoir 604 at the inlet / outlet, and then the liquid reservoir 604 is filled. Affix the sticker. The user first removes this seal and overlays an appropriate amount of specimen on the inlet. If this is the case, the sample can be easily set by a normal pipette operation, and air bubbles are not mixed at the interface between the buffer and the sample that communicates in the flow path, so that the possibility of failure in separation can be eliminated. The buffer that fills the flow path in advance has a density close to the specific gravity of the target particle in order to prevent sedimentation of particles contained in the sample in addition to physiological saline used for the purpose of diluting the assumed sample. A liquid etc. can be considered.

  FIG. 20 shows an example in which a chip having the cross-sectional structure shown in FIG. A flow path pattern 2002 is formed on the surface of the flow path carrier 2001, and a closed flow path is formed by joining a plate 2003 to this surface. As the introduction port of the flow path, in addition to the sample liquid reservoir 2004, as described above, a buffer liquid reservoir 2005 for pressing particles to the branch flow path side is provided as necessary. A collection liquid reservoir 2006 is provided at the outlet downstream of the branch channel group, and is collected separately for each separation size. Since particles larger than any separation size do not enter the branch flow path, they are recovered from the non-branch recovery liquid reservoir 2007 ahead of the main flow path. When the number of entrances / exits is large, the flow path carrier 2101 may be formed in a block shape as shown in FIG. 21, and the structure may be provided with the inlet port 2102 and the recovery port 2103 to the flow path pattern.

  After setting the specimen with the configuration as shown in FIG. 6, how to realize liquid feeding to the flow path is the next issue. The most natural method is to connect a tube to the liquid reservoir 604 and pressurize with a compressor or a pump. FIG. 7 shows an example of the configuration. The microchannel chip used in the system of the present invention is formed by joining the plate 702 to the channel carrier 701 as before. A cylindrical liquid reservoir 703 is formed at the inlet of the chip, and the specimen 704 is set in a layered manner on the buffer liquid surface filled to the bottom. From this state, first, a tube 705 is connected to the liquid reservoir 703, and the other end of the tube 705 is connected to a tip side pipe 707 provided so as to protrude from the pressurized tank 706. A valve 708 is installed in the chip side pipe 707, and pressurization to the chip can be started or stopped as necessary. In addition, a pressure adjustment valve (regulator) 709 is also installed, and the pressure applied to the chip side can be adjusted. On the other hand, the pressure source side pipe 710 is connected with a pressurizer 711 such as a gas cylinder, a pressurizing pump, and a compressor, and thereby the inside of the pressurizing tank 706 is pressurized. If a gas cylinder is used as the pressurizer 711, the system of the present invention can be used without requiring electric power.

When the pressurized tank 706 does not require a large capacity, the pressurizer 711 can be pressurized by manually pushing the piston, such as a syringe or a bicycle inflator, and can be fixed in that state. Can be used. If a compressor or the like is used as the pressurizer 711, strict pressure control is possible. The pressure tank 706 is also provided with a pressure gauge 712. Normally, the pressure adjustment valve 709 is adjusted while checking the pressure to control the pressure applied to the chip, that is, the liquid feeding speed to the chip. However, if the pressure gauge 712 is capable of electrical output, the pressure can be automatically adjusted by feeding back the output.
In particular, as a method of pressurization when forming a laminar flow in the main flow path on the flow path carrier 701, for example, when the cross section of the main flow path is 100 μm × 50 μm, the flow rate per hour is 0.2 ml / h to 3.0 ml. The flow rate is adjusted to about / h.

  FIG. 8 shows another form of a liquid feeding system using gas pressurization. The microchannel chip is formed by joining a plate 802 to a channel carrier 801 as before. Here, the sample 803 is set in the liquid reservoir at the inlet portion of the chip, and then the chip is accommodated in the pressurized tank 804. The pressurization tank 804 is pressurized by the pressurizer through the pressure source side pipe 805 as in the example of FIG. Further, the pressurized tank 804 is provided with a vent 806. By connecting the tip outlet 807 and the vent 806 with a tube or the like, the outlet side of the tip is exposed to atmospheric pressure. Even if nothing is connected to the inlet side of the chip, it is pressurized in the pressurized tank 804, so that a pressure difference is generated inside the chip and liquid feeding can be executed. It is assumed that the pressurized tank 804 is provided with a mechanism capable of opening or closely fixing the upper part in order to insert and remove chips. When a plurality of chips are used at the same time, the pressurized tank 804 may be provided with a plurality of vent holes 806 corresponding to the maximum number of simultaneously used chip outlets, and each chip may be accommodated in the pressurized tank 804. . Alternatively, prepare multiple pressurization tanks 804 and connect to each pressurizer, or prepare separate pressurization tanks to distribute the same pressure to multiple pressurization tanks, and connect the pressurizer to this distribution pressurization tank May be.

  FIG. 9 shows still another embodiment of a liquid feeding system using gas pressurization. As in the previous examples, the microchannel chip 901 is installed in the pressurized tank 902, the sample is set in the liquid reservoir 903 on the inlet side, and the pressurized tank is closed for use. The outlet 904 is formed on a surface (back surface) opposite to the inlet. A drainage port 905 is opened in the pressurized tank 902, and the sample after separation can be collected from this by aligning the position with the tip outlet 904. However, if gas in the pressurization tank leaks through the drainage port 905, pressurization cannot be performed. Therefore, the chip is fixed by sandwiching an elastic packing material 906 between the back surface of the chip and the pressurization tank. If the packing material 906 is attached to the back surface of the chip from the beginning, the operation becomes simple.

When handling biological specimens, there is a strong demand to avoid contact with specimens as much as possible. Therefore, it is preferable that the packing material 906 has a shape that can cover the inner surface of the drainage port 905 of the pressurized tank as shown in FIG. With such a structure, it is not necessary to connect a tube or the like at both the inlet and outlet of the chip, and it becomes easier to handle the chip. The pressurized tank 902 needs to be structured so that the sample collection container 907 can be installed on the bottom side. In the example of FIG. 9, the outlet is schematically illustrated as one, but when there are a plurality of outlets, a plurality of drainage ports 905 are opened, and the sample collection container 907 is also provided with a partition inside, It is preferable to install a plurality of small sample collection tubes. Since the sample collection container 907 is not limited by the chip size, it is particularly suitable when it is desired to obtain a large amount of collection by long-time separation.
If the sample can be collected from the back surface of the chip, the above-described advantages can be obtained. Therefore, if the sample collection container 1001 can be directly fixed to the back surface side of the chip as shown in FIG. Whether connecting a tube or installing the entire chip in a pressurized tank, both can be accommodated.

  Further, as shown in FIG. 11, a configuration in which a liquid reservoir 1102 at the chip inlet is provided on the chip plate 1101 side and an outlet 1104 is provided on the flow path carrier 1103 side is also useful. If the chip is arranged in the pressurized tank as shown in FIG. 11, the microchannel carrier 1103 is not affected by the pressurization. In particular, when the microchannel carrier 1103 is formed of an elastic material such as PDMS, there is a concern that the channel may be deformed by pressurization, but this problem can be solved. Note that the packing material 1105 for preventing leakage of gas in the tank is necessary.

  When the amount of specimen to be processed by the system of the present invention is relatively large, for example, exceeding 10 times the chip capacity, the configuration shown in FIG. 12 is also useful. That is, the specimen 1201 is injected into the specimen container 1202 and set inside the pressurized tank 1203. The sample container 1202 and the inlet 1205 of the microchannel chip are connected via a connection tube 1204 such as a hose or a tube. If the connection relay port 1206 is provided in the pressure tank 1203, the connection pipe from the sample side and the chip side can be separately connected to the pressure tank 1203, so that the chip can be easily arranged outside the pressure tank. If pressure is applied to the pressure tank 1203 in this state, liquid feeding can be easily realized. Since the sample container 1202 is completely independent of the microchannel chip, the capacity of the sample container 1202 is not limited by the size of the chip. Therefore, the configuration of FIG. 12 is useful when the amount of the sample is large.

  In addition, when the particle size of the specimen to be processed by the system of the present invention is relatively small, such as 1 μm or less, and the channel resistance of the microchannel chip must be increased, the configuration as shown in FIG. 13 is useful. . That is, only the liquid reservoir 1302 at the inlet of the microchannel chip 1301 is set inside the pressurized tank 1303, thereby limiting the area of pressure necessary for liquid feeding to the cross-sectional area of the liquid reservoir 1302. To do. As a result, the volume of the pressurized tank 1303 can be reduced. Therefore, even when a large pressure is required according to the flow path resistance, the pressure tank as a whole does not need to be so large and can be used safely. It becomes easier. Since there is no pressurization to the chip, it is not affected by the deformation of the flow path due to the large pressurization.

  In such a configuration, the packing 1304 for preventing gas leakage from the pressurized tank 1303 is inserted around the liquid reservoir 1302 which is a contact portion. In the shape shown in FIG. 13, it is impossible to pass the liquid reservoir 1302 through the circular hole. Therefore, the liquid reservoir 1302 is sandwiched and fixed by a structure having two semicircular cutouts. is required. As shown in FIG. 14, as a method of pressurizing only the liquid reservoir 1402 at the inlet of the microchannel chip 1401, an elastic body 1403 that is in close contact with the inner surface of the liquid reservoir 1402 is used to apply pressure by pushing like a syringe. However, if there is such a mechanical contact, a large force is applied to the chip itself, which may cause deformation, and it is difficult to stabilize the pressure only with the capacity of the liquid reservoir 1402. It's not a very appropriate method.

When the microchannel chip and the fluid drive unit used in the present invention described in the fifth embodiment are summarized, the description is as shown in 1 to 10.
1. A fluid drive device for a micro flow path formed by a drive means for feeding liquid to a chip by using an indirect pressurization to a flow path by an inert gas or indirectly through a space.
2. It has a cylindrical liquid reservoir structure at the inlet of the chip, and the sample can be easily supplied to the flow path by injecting the sample into the liquid reservoir structure and then pressurizing the liquid reservoir structure. A driving means is proposed.
3. A combination of a chip and a driving means in which there are a plurality of inlets of the chip, and each inlet is pressurized directly from the same pressure source through a cylindrical liquid reservoir or indirectly through a sealed space. A configuration is proposed.
4). A combined configuration with driving means for performing liquid feeding by connecting a tube from a pressure source to the liquid reservoir structure at the inlet of the chip;
5. A combined configuration with a driving means for accommodating the chip in a pressurized container and indirectly pressing the chip;

6). A configuration for discharging the liquid to the outside by connecting the outlet of the chip to a vent provided in the pressurized container,
7). 7. Chip configuration in which the inlet of the chip is formed on the top surface and the outlet is formed on the back surface. A chip configuration that allows the outlet of the chip formed on the back surface to coincide with the position of the drainage port formed on the bottom surface of the pressurized container, and allows the sample to be collected from the drainage port;
9. By providing the chip used in the system with the buffer solution filled in advance, there is no need to wait for the disappearance of the air remaining in the flow path, or to the interface between the sample and the liquid leading to the inside of the chip. A configuration that can prevent air contamination
10. A configuration that enables the precipitation of particles in the chip to be prevented by preliminarily filling the chip with a liquid having a density comparable to the specific gravity of the particles for separation and recovery.

As described above, the advantages of the liquid feeding system using the pressurization by gas compared with the flow rate control type liquid feeding device such as a syringe pump are as follows.
The first advantage is that the use of the system can be expanded because the system can be used even in places where there is no power. For example, it is possible to collect river water and inspect it on the spot, or to investigate the infection situation at the disaster site on the spot.
The second advantage is that liquid feeding without pulsation becomes possible. In the case of controlling the liquid feeding speed using a stepping motor like a syringe pump, pulsation occurs when the rotational speed is lowered. As a result, laminar turbulence may occur locally, or the instantaneous balance of the amount of liquid fed from a plurality of inlets may be lost. This is fatal because the physical principle does not hold for the system of the present invention. In the case of pressurization with gas, pulsation does not occur in principle, and even if multiple inlets are connected to the same pressurization tank, the same liquid feeding force is always applied. There is no problem and stable liquid feeding is possible. Since it is easy to use not only a plurality of inlets but also a plurality of chips at the same time, it is very effective when a large amount of samples are to be processed in a short time.

The third advantage is that the specimen can be easily set and there is no waste. As already described, when a sample is sent to a chip using a tube, bubbles are easily mixed when the tube is connected, and the sample remaining in the tube is wasted. The latter is a fatal problem, especially when only a small amount of specimen can be prepared.
Since the liquid feeding system using the pressurization by the gas as described above can be used for all devices such as chips that require liquid feeding, the micro flow path using the laminar flow used in the system of the present invention. The application is not limited to the chip. In addition, the pressure at the time of pressurization used with the system of this invention is the range of 1.1-2.5 atmospheres, for example. As the gas, an inert gas such as nitrogen that does not affect the specimen is mainly used, but oxygen, carbon dioxide, air, etc. are also used as appropriate if it is assumed that the cells contained in the specimen remain alive. The Further, the above-described effects due to pressurization to the chip inlet side can be similarly realized by reducing the pressure on the chip outlet side. For example, a method in which a chip (at least an outlet portion) is placed in a decompression tank instead of being placed in a pressurized tank, and suction is performed by connecting a tube to the outlet side is essentially equivalent.

When the present invention is applied to uses such as clinical examination or industrial product classification, it is more convenient to automate the entire system. An example of an automated system is shown in FIG.
The automation system 1501 of the present invention includes a stage 1503 for installing the microchannel chip 1502 and a liquid feeding device 1504. When a fixed-quantity liquid feeding device driven by a motor such as a general syringe pump is incorporated as a liquid feeding device, the micro-channel chip 1502 is connected by a tube 1505, and the specimen is introduced into the chip through the tube 1505. . As already mentioned, such a liquid delivery device has a problem that a dead volume that remains in the tube is large and a pulsating flow is likely to occur. But you can. In this case, a pressurized tank as described in the fifth embodiment is incorporated in the system. Note that a gas cylinder or a pump serving as a pressurizing source does not need to be incorporated in the housing of the automation system 1501, and may be connected from the outside. When a gas pressurization system is used, the connection with the tube 1505 is complicated, and therefore, after setting the specimen in the liquid reservoir at the inlet of the microchannel chip 1502, the microchannel chip 1502 is added to the automation system 1501. A system that can be used if installed in a pressure tank is preferred. As a structure, a pressurization system as shown in FIGS. 8 to 14 is incorporated in the system.

  What is important as an automated system is that the state of the microchannel chip 1502 can be monitored. The monitoring device 1506 is preferably a camera, an image sensor, a photodiode array, or the like. This is because the microchannel pattern used in the present invention has a very large number of channels, and it is more suitable to monitor the whole as an image rather than to observe the fixed point. Thereby, for example, it is possible to perform automatic control such as detecting that bubbles are present in the flow path and notifying them, and waiting for liquid feeding of the specimen until the bubbles disappear with the liquid feeding only as a buffer. Even after the liquid feeding of the specimen is started, it is possible to detect that a part of the flow path is clogged with a large impurity and stop the notification and liquid feeding. In addition, by counting particles passing through a plurality of channels by image analysis, it is possible to count particles at a much higher speed than a particle counter using a single channel. Cell sorter systems that do not require fluorescent labeling or cell sorter systems that use more detailed features can be realized by controlling the shape of particles by image analysis and switching the flow path branch destination according to the particle shape. Will do. Note that a monitoring apparatus that can obtain an image may not necessarily be used as long as it is configured to use a plurality of optical fibers and simultaneously monitor a plurality of portions of the flow path pattern. In addition, since the microchannel pattern used in the present invention can change the size range to be separated and recovered stepwise (continuously), it is also possible to measure the size distribution of particles in the specimen. . Such products are distributed as particle size distribution analyzers, but if you have a function to count the number of particles going to each outlet of the microchannel chip whose separation size range has been changed in stages, the same processing It can be performed.

  When processing is performed inside the system as shown in FIG. 15, a detection light source 1507 is also necessary. When monitoring an image, a white light source is desirable, but when a particle contained in a specimen or a label thereof emits fluorescence, a light source such as a laser or a light emitting diode having an excitation wavelength suitable for fluorescence may be used. In addition to the monitoring device 1506, a sensor 1508 may be installed as necessary. Examples of the sensor 1508 include a photodiode, a photomultiplier tube, a magnetic sensor, a capacitance sensor, and an ultrasonic sensor.

In the detection by the monitoring device or sensor, the count of dilute particles is particularly significant. As described above, the microchannel chip used in the system of the present invention is very useful when the existence frequency is very low, that is, when the purpose is to collect dilute particles. In particular, when it is desired to collect a certain number of dilute particles instead of the amount of specimen, the amount of specimen necessary for that purpose is indefinite at the start of processing, and therefore the time required for processing is unknown. For example, this is the case when a certain number of cells need to be collected when the purpose is to perform genetic diagnosis on a specific cell or when the purpose is to reliably culture a specific cell. . If the site where the target particles are captured or stored is monitored, a notification is given when the required number is reached, and the process can proceed to the next process. In addition, even if it is possible to make a judgment if the number of specific particles contained in a certain amount of sample exceeds the specified value, the specified amount of sample exceeded the specified value before reaching the specified amount. If it can be confirmed by monitoring, quicker judgment will be possible.

For example, it can be applied to a diagnosis that tumor metastasis is observed if the number of circulating tumor cells in the blood exceeds a specified value. Such monitoring is not limited to the state immediately after separation by the chip, but can be implemented as a detection method in which the signal is amplified with the passage of time, such as gene amplification (PCR) method, cell culture, and enzyme reaction inside the chip. If it is, the same effect can be obtained if the site is monitored. Chips that use laminar flow separation can concentrate and collect fractions in a specific size range, so combining these detection methods with concentration also has the effect of obtaining signals more quickly. I can expect. In addition, by having means for supplementing circulating tumor cells with multiple types of antibodies, the type of cell is specified by the position where the antibody is immobilized or the fluorescent color of the fluorescent particles bound to the antibody, etc. There may be provided means for not only counting but also determining a site where a tumor has occurred.

More specifically, the present invention can show a combined configuration of a blood particle classifying means for tumor cells in blood and a tumor cell detecting means for performing an antigen-antibody reaction and a reagent reaction.
The blood particle classifying means extracts a particle group in the size range of tumor cells in a combination of a plurality of branch channel groups and a main channel. For example, in the trap region 1903 shown in FIG. A membrane on which an antibody that binds to a tumor cell surface antigen is immobilized is placed, the tumor cells that pass through the trap region 1903 are captured, and then the free antibody to which the above-described label is bound is supplied to the trap region 1903, followed by washing. A method of measuring the presence or absence of a label in the later trap region 1903,
An antibody with a label that specifically binds to tumor cells may be supplied to the blood in advance and supplied from the input unit 1900a.
At that time, the classification operation may be performed by introducing a buffer from the buffer introduction portion 1900b formed in order to positively perform the classification operation to the branch channel group 1902.

In addition, in order to detect unspecified tumor cells, a plurality of types of antibodies that specifically bind to an assumed tumor cell membrane are prepared, and these are fixed to the trap region 1903 for each type of antibody, and unspecified. Presence of tumor cells is detected, or antibodies that are previously bound to a plurality of different types of labels are supplied to the specimen, and then a different region is set for each antibody in each of the trap regions 1903, and a doctor diagnoses cancer It may be a thing.
Although the trap region 1903 is arranged on the branch channel group 1902 side, the trap region 1903 is not limited to this and may be provided in the main channel.
Since the branch channel group 1902 in this case is for removing unnecessary blood cells and plasma by branching, the trap region 1903 is preferably provided after that.
In this example, the trapping region 1903 is configured to capture target tumor cells or unspecified tumor cells. However, the trapping region 1903 is simply supplied with a label bound to a free antibody, and classified according to particle size. The particle group in the range including tumor cells may be collected and observed by the output unit 1900c (separation collection unit 1901) and separation collection units 1901 and 1904, or the presence or absence of the label may be observed on the spot. . In that case, different types of tumor cells can be detected by an observation device by binding a different label (fluorescence wavelength, dye, etc.) for each tumor to the antibody.

FIG. 19B shows a specific example of the trap region 1903.
Reference numeral 1903a denotes a magnetic member, which is composed of a permanent magnet or an electromagnet, and is disposed outside the flow path. The magnetic member 1903a is not limited to this, and may be, for example, for forming a DC electric field with an electrode pair or for forming an alternating electric field. is there.
Reference numeral 1903b denotes a supplemental cell storage unit that temporarily stores nucleated cells, tumor cells, and other rare cells, and is provided with a step or the like that is lowered so as not to move in the direction of the main flow path.
Reference numeral 1903c is a supplemental cell, which is a cell intended to supplement nucleated red blood cells, tumor cells and the like. 1903d is a label, and examples thereof include Dynabeads (manufactured by Invitrogen) and MACSbeads (manufactured by Miltenyi Biotech). Here, magnetic particles of the S pole are shown. The label 1903d is bound to an antibody that specifically binds to the antigenic site of the supplemental cell.
The operation of the configuration shown in FIG. In the trap region 1903 or the region in front of it, the south pole magnetic particles are bound to the supplementary cell 1903c via an antibody.
The supplemental cells 1903c combined with the label 1903d change in the moving direction under the influence of the magnetic field of the magnetic member 1903a and are stored in the supplemental cell storage unit 1903b.
In addition, when the magnetic field of the magnetic member 1903a is strong or when the flow is slow, the magnetic member 1903a may be supplemented without moving to the supplemental cell reservoir 1903b.

The present invention is an effective judgment for detection of tumor cells, and also functions effectively for detection of tumor markers generated in blood in relation to tumors. In the case of a tumor marker, for example, as shown in FIG. 1, it specifically binds to a liquid collection channel group 13a, 13b, which is a site for collecting plasma, and a coloring reagent for a tumor marker or a tumor marker antigen. It can be detected by a combination with the immunological test strip A18 and the immunological test strip B19 equipped with the antibody to be tested.
Alternatively, a tumor marker reagent or the above-described antibody may be fixedly disposed in the main channel after separating and removing all the particles in the branch channel.
In the case of an antibody, a separate free antibody labeled with fluorescence, magnetism or the like is prepared, and the labeled free antibody is further bound to a tumor marker bound to the immobilized antibody, and the label is measured. It may be possible.

For specimens that can be judged if a certain number or more of particles are counted as described above, the separation operation was performed normally only by the fact that the particle count was less than a certain number. Since there is no guarantee, questions remain about the accuracy of the judgment. Therefore, it is preferable to mix the marker particles separated in the same size as the particles to be counted in advance in the specimen, and to count not only the target particles but also the marker particles with respect to the separated components after separation. .
An active device 1509 that performs some action on the chip is also used as necessary. For example, a magnetic field generator such as an electromagnet is operated to move particles or magnetic beads bonded to the particles in the flow path, an electric field is applied from the electrode to the chip to move the particles by dielectrophoresis, a heater or For example, the heat of the laser is applied to the chip to deform the flow path in the chip, and the entire chip is kept at a constant temperature by a heater to prevent the sample from being altered. In addition, regarding the heat retention or cooling of the specimen, the temperature of the gas used for pressurization or the temperature of the pressurization tank may be controlled by a heater or the like. The specimen processed by the microchannel chip 1502 in this way is collected from each outlet of the chip. It is desirable that the sample container 1510 is set in the system so that the collected material from the outlet can be stored. When there are a plurality of outlets, the inside of the sample container 1510 is also divided into a plurality of regions by a partition so as not to be mixed. Alternatively, the specimen container 1510 may have a shape in which a hole is provided at a position corresponding to each outlet so that a commonly used sample tube can be set.

  As described above, a flow cytometer is generally used as a measuring instrument for obtaining information on cells contained in a specimen. This can also be realized in the system according to the present invention by devising the structure. For example, as shown in FIG. 22, if the buffer introduction unit 2202 is arranged so as to sandwich the sample introduction unit 2201, the sample flow 2203 is not pushed to one side as in the previous examples, but the central part of the main channel 2204. Get together. In a general flow cytometer, the flow of cells is narrowed in the center and the cells are detected with a laser beam or the like, so that the same detection can be performed. As the driving force for feeding the sample and the buffer, if a structure in which the same pressure is applied to each inlet port as described above is used, stable liquid feeding is possible. If it is desired to perform the same size separation as before after detection, it is necessary to press the specimen flow 2203 to the branch flow path side, but this adds a buffer introduction part to the main flow path one side downstream of the detection part. Alternatively, this can be realized by providing a buffer branch channel group 2205 for absorbing the buffer flow on one side. Note that size separation may be performed first, and a buffer introduction portion as shown in FIG. 22 may be provided downstream of the branch flow channel group so that the sample flow is arranged at the center of the flow channel. In this case, since detection is performed only on particles that have already been separated into a specific size range, when it is desired to obtain information only about the range, the detection efficiency can be dramatically improved.

In addition to the above, functions that can be used as appropriate in an automated system include, in addition to the above, a function that automatically dispenses a specimen from a predetermined specimen installation region to the inlet of the microchannel chip, after the use of the microchannel chip. The function of feeding a buffer solution for washing the inside of the microchannel, the function of picking up specific particles (cells) from the sample collected from the outlet of the microchannel chip, and the reservoir part in which the specimen is stored Or the function which shakes or stirs the whole microchannel chip | tip, the discard of the sample collect | recovered from a chip | tip while the initial laminar flow state is unstable, etc. can be mentioned.

The chip and driving means according to the present invention described in this embodiment and the monitoring means for monitoring the fluid and particles on the chip are summarized as follows.
1. 1. A structure in which a gas cylinder or a pump serving as a pressurizing source for supplying liquid is connected to a chip installed in the system. 2. Monitor means such as a camera for monitoring a plurality of flow path states in the chip with respect to the chip installed in the system. The plurality of flow path states in the chip monitored by the monitoring means include one or more of the presence or absence of bubbles, the occurrence of clogging of the flow path, the number of passing particles, and the shape of passing particles.

4). The means for monitoring a plurality of flow path states in the chip has means for acquiring the image information of the flow path as a moving image or a still image.
5. It has a notifying means for monitoring the number of collected particles designated in advance and notifying that the number of particles has reached a preset value by means of screen display, voice display or the like.
6). An automatic processing means is provided for monitoring the number of collected particles designated in advance and automatically executing the designated processing on the particles when the number of particles reaches a preset value.

  The greatest feature of the method for controlling the behavior of particles in the fluid in the system of the present invention is that the main flow path and the branch flow path are already described in this specification or in a document such as Japanese Patent Application Laid-Open No. 2007-175684. By appropriately designing the flow path resistance ratio, the flow rates in these directions are accurately controlled. This determines the width of the laminar flow on the branch channel side, and whether or not the particles in the fluid can travel in the direction of the branch channel is the size that the particle (the center of gravity) puts in the laminar flow on the branch channel side Is determined by whether or not. This principle will be described with reference to FIG. 16. When the fluid traveling in the main flow path 1601 reaches the branch point 1603 between the main flow path 1601 and the branch flow path 1602, the flow of the main flow path 1601 ′ after the branch point 1603 is flown. Based on the ratio between the channel resistance value and the channel resistance value of the branch channel 1602, the flow rate traveling in each direction is determined. The flow distribution is a quadratic function that is 0 at the wall surface and maximum at the center of the flow path in accordance with the hydrodynamic principle. Therefore, when the flow rate is determined, the width W of the laminar flow on the branch flow path side is automatically determined. That is.

  Note that the flow rate that travels straight through the main flow channel and the flow rate that travels toward the separation flow channel are determined simply by the ratio of the flow channel resistance values if the pressure at the end of each flow channel is equal. Usually, the end of any flow path is an open end, and the pressure at the end is at atmospheric pressure, so this precondition is satisfied. However, when pressurizing or depressurizing any terminal (exit), each flow rate is obtained by dividing the pressure gradient between the branch point 1603 and each terminal by the channel resistance value. The flow rate of each flow path, that is, the width of the laminar flow is not determined only by the ratio of the values. If this principle is positively utilized, for example, by applying pressure to the outlet on the branch flow path side, the flow rate on the branch flow path side can be further reduced and the width of the laminar flow can be reduced. The same phenomenon occurs even if the flow rate on the main channel side is increased by reducing the pressure at the outlet on the main channel side. When it is desired to separate only very small particles such as 1 μm or less on the branch channel side, it is necessary to reduce the flow rate on the branch channel side to suppress the laminar flow width W to about 0.5 μm. If an attempt is made to increase the resistance only by increasing the path resistance value, the flow path width of the branch flow path must be narrowed or lengthened, resulting in a problem that the flow path is easily clogged or the entire chip is enlarged.

Therefore, the method of controlling the width of the laminar flow by controlling the pressure on the outlet side of the flow path is very effective. If the pressure on the outlet side is appropriately changed, a plurality of laminar flow widths can be realized with the same flow path pattern, and the proper use according to the purpose is also possible. If the pressure reduction on the outlet side is performed by attaching a vacuum container to the outlet, the separated specimen can be recovered by this vacuum container. As a method of changing the flow path resistance value, a part of the flow path pattern is locally heated and deformed with a laser beam or the like, a part is attached to or detached from the flow path pattern, and an electric field or magnetic field is applied to the flow path pattern. A method of deforming, bending the branch flow path, or providing a column structure can be used as appropriate.

  The present invention is a chip with a diagnostic function having the function of separating and supplying blood, for example, according to the flow. In addition, there are a wide variety of such things as antibody production, environmental water analysis, food poisoning related microbiological tests, infectious disease diagnosis, etc. Therefore, it can be applied to mobile diagnostic chips such as POC, infectious disease testing chips, immune chips, disease diagnostic chips such as periodontal disease, environmental testing chips, standard ball sorting devices, and the like.

DESCRIPTION OF SYMBOLS 1 Body fluid type | system | group solution input part 2 Separation extraction part 3 Analysis operation means 4 Drive means 5 Carrier 1a Supply part 1b Diluent supply part 1c Output destination 501 Specimen introduction part 502 Main flow path 503 Branch flow path group 504 Vent 505 Plasma recovery part 601 Channel carrier 602 Channel pattern 603 Plate 604 Liquid reservoir 605 Liquid level 701 Channel carrier 702 Plate 703 Liquid reservoir 704 Sample 705 Tube 706 Pressure tank 707 Chip side piping 708 Valve 709 Pressure adjustment valve 710 Pressure source side piping 711 Pressurizer 712 Pressure gauge

Claims (47)

A component adjustment input unit that adjusts and inputs biological components such as body fluid solutions,
A separation / extraction unit for separating and extracting a target component or liquid in the adjustment liquid adjusted and input to the component adjustment input unit, for analyzing and operating particles or liquid obtained in the separation / extraction unit and outputting related information A carrier comprising an analysis operation means, wherein each part and each means of the carrier are connected by a flow path, and the flow path is a biological operation in which the biological component is moved by a flow generated by a drive means. system. 2. The biological flow according to claim 1, wherein the flow generated by the driving means is one of the laminar flow and the flow obtained by changing the flow direction of the branched flow path in a direction selected according to the characteristics of the particles. Operation system. The separation and extraction unit includes a flow path A having a size that does not cause the particles to branch in the moved fluid, and a flow path B that branches the particles having a target size depending on the size of the particles in the fluid. The biological manipulation system according to claim 1 provided. The biological operation system according to claim 1, wherein the analysis operation means provides immunodiagnosis information and allergy information. The biological manipulation system according to claim 1, wherein the analysis manipulation means is an immunization production step. The biological operation system according to claim 1, wherein the analysis operation means outputs related information such as health / disease related information, environmental water, and water and sewage. The biological operation system according to claim 1, wherein the analysis operation means outputs diagnostic information such as tumor marker information and virus infection. The biological operation system according to claim 1, wherein the analysis operation means outputs lifestyle-related disease diagnosis information such as diabetes / arteriosclerosis-related disease. The biological manipulation system according to claim 1, wherein the information output by the analysis manipulation means is direct information that can be directly diagnosed, or indirect information that is used for a plurality of examinations, such as secondary and tertiary. The information is color development information, movement position information, fluorescence information, size information, other shape information, sequence information, state information such as presence / absence of binding, immunoprecipitation information generated in the state of binding to beads, presence / absence information of sedimentation, Information on presence / absence, strength, polarity, etc. of electric charge, information on presence / absence of magnetism, strength, polarity, etc., concentration information, temperature information, viscosity information, strain information. A biological manipulation system as described in. An adjustment input unit that adjusts and inputs a medium including a sphere, a separation and extraction unit that separates and extracts a target sphere in the adjustment medium that is input to and adjusted by the adjustment input unit, and a sphere obtained by the separation and extraction unit is analyzed and operated. A carrier having analysis operation means for outputting sphere specifying information, wherein each part and each means of the carrier are connected by a flow path, and the flow path is operated by a flow generated by the drive means. Industrial operation system. 12. The industrial flow according to claim 11, wherein the flow generated by the driving means is one of the laminar flow and the flow in which the direction of moving the branched flow path in the direction selected by the characteristics of the spherical particles is changed. Operation system. It has a structure in which there is a branch flow channel connected to the side of the main flow channel, and the flow width traveling to the branch flow channel side is determined by controlling each flow rate traveling to the main flow channel and the branch flow channel. Biological and industrial operating systems characterized. 14. The biological operation according to claim 13, wherein the means for controlling each flow rate proceeding to the main channel and the branch channel is based on a ratio of channel resistance values of the main channel and the branch channel. System and industrial operation system. The separation threshold based on the particle size can be set smaller than the width of the branch flow path by making the width of the flow traveling toward the branch flow path smaller than the width of the branch flow path. The biological operation system and industrial operation system of 13 and 14. The biological operation system and the industrial operation system according to claim 15, wherein the width of the flow traveling toward the branch channel is equal to or less than half the width of the branch channel. The number of the branch channels is in the range of logn to 20 logn (log is a logarithm with 10 as a base) with respect to the number n of particles per 1 ml of the sample for separation and recovery. The biological operation system and industrial operation system of Claims 13-16. Biological operating system and industrial operation having a function of eliminating particles having a size smaller than the target particle by the first-stage branching channel group disposed on the side of the main channel upstream of the main channel system. A function having a function of separating particles in a size range including target particles by a branch channel group of the second and subsequent stages disposed downstream of the main channel from the branch channel group of the first stage. Item 19. The biological manipulation system and the industrial manipulation system according to Item 18. The biological operation system and the industrial operation system according to claim 18 or 19, wherein the size separation function of the branch channel group is realized by controlling a width of a flow of a liquid traveling to the branch channel side. . The separation size range of the first-stage branch channel group is set to (3-α) μm or less (where α is any value satisfying 0 ≦ α ≦ 2), so that the specimen is blood. 21. The biological operation system and the industrial operation system according to claim 18, wherein only the plasma component not containing blood cells is recovered through the first-stage branch flow path group. When the separation size range of the n-th branch channel group is set to (7−α) μm or less (where α is any value satisfying 0 ≦ α ≦ 2), The biological operation system and the industrial operation system according to claim 18 to 21, wherein red blood cells in the sample are excluded through the n-th stage branch channel group. Red blood cells are eliminated by the n-th branch channel group, and the separation size range of the (n + 1) -th branch channel group disposed downstream of the n-th branch channel group with respect to the main channel. Is set to (10 + α) μm or less (where α is any value satisfying 0 ≦ α ≦ 4), so that a group of particles mainly composed of white blood cells not containing red blood cells becomes the (n + 1) -th branch channel. 23. The biological and industrial operating system of claim 22 that is excluded through the group. The separation size range of the n-th branch channel group is set to (10 + α) μm or less (where α is any value that satisfies 0 ≦ α ≦ 4), and is located downstream of the branch channel group from the main channel. By setting the separation size range of the arranged (n + 1) -th branch flow path group to (20 + α) μm or less (where α is any value satisfying 0 ≦ α ≦ 5), In some cases, particles such as stem cells, circulating tumor cells, iPS cells, and various cell masses that are larger than leukocytes on average can be collected through the branch channel group. Operation system and industrial operation system. For a liquid sample containing a plurality of types of particle groups, a means for separating the particle groups according to the difference in particle size, and a fraction containing the particle groups limited to the same size range obtained through the means A biological operation system and an industrial operation system having a function of supplementing target particles using properties other than size among components, or having a function of capturing other than target particles. The means for separating the particle group according to the difference in the particle size is the organism realized by advancing a part of the liquid sample traveling in the main channel to the branch channel connected to the main channel. Operation system and industrial operation system. 27. The biological manipulation system and the industrial manipulation system according to claim 25, wherein the means for capturing the particles is by using an antigen-antibody reaction. Biological operation system and industrial operation system having means for extracting nucleic acid in cells from cells collected at a specific site on the chip by size separation, and a function of amplifying nucleic acid using polymerase chain reaction . A biological operation system and an industrial operation system having a function capable of culturing cells collected on a specific site on a chip by size separation on the chip. A biological operating system and an industrial operating system, wherein liquid is fed to a chip using pressurization by gas. 31. The biology according to claim 30, wherein the tip has a cylindrical liquid reservoir structure, and a specimen is injected into the liquid reservoir structure, and then pressurized to the liquid reservoir structure. Operation system and industrial operation system. 32. The biological operation system and the industrial operation system according to claim 30, wherein there are a plurality of inlets of the chip, and each inlet is pressurized from the same pressure source. 33. The biological operation system and the industrial operation system according to claim 30 to 32, wherein liquid feeding is executed by connecting a tube from a pressure source to a liquid reservoir structure at the inlet of the chip. The biological operation system and the industrial operation system according to claim 30, wherein the chip is accommodated in a pressurized container and pressurized. The biological operation system and the industrial operation system according to claim 34, wherein an outlet of the chip is connected to a vent provided in a pressurized container. The biological operation system and the industrial operation system according to claim 34, wherein an inlet of the chip is formed on an upper surface and an outlet is formed on a rear surface. 37. The biology according to claim 36, wherein the outlet of the chip formed on the back surface and the position of the drainage port formed on the bottom surface of the pressurized container are matched, and the sample is collected from the drainage port. Operation system and industrial operation system. A biological operation system and an industrial operation system, characterized in that a chip used in the system is provided in a state in which physiological saline is filled in advance. A biological operation system and an industrial operation system, characterized in that a chip used in the system is provided in a state where a liquid having a density comparable to the specific gravity of particles intended for separation and recovery is filled in advance. A biological operation system and an industrial operation system having a structure in which a gas cylinder or a pump serving as a pressurizing source for supplying a liquid is connected to a chip installed in the system. A biological operation system and an industrial operation system having a function of monitoring a plurality of flow path states in a chip installed in the system. 42. The plurality of flow path states in the chip to be monitored include any one of presence / absence of bubbles, occurrence of clogging of the flow path, the number of passing particles, and the shape of passing particles. Biological and industrial operating systems as described. 43. The biological operation system and the industrial operation system according to claim 41, wherein the means for monitoring a plurality of flow path states in the chip acquires image information of the flow path. A biological operation system and an industrial operation system having means for monitoring the number of particles collected in advance and notifying that the number of particles has reached a preset value. The number of collected particles specified in advance is monitored, and when the number of particles reaches a preset value, a specified process is automatically executed on the particles. Operation system and industrial operation system.   2. The biological manipulation system according to claim 1, wherein the analysis operation means detects a specific tumor cell by arranging an antigen or an antibody that specifically binds to the specific tumor cell in a free or fixed manner. Industrial operation system. 2. The biology according to claim 1, wherein the analysis operation means can unconditionally diagnose tumor cells by arranging a plurality of antigens or antibodies that specifically bind to the tumor cells in a free or fixed manner. Operation system and industrial operation system.

Images