JP5158111B2 - Reaction analysis method - Google Patents

Description

  The present invention relates to a reaction analysis method in a flow path disposed on a substrate. More specifically, the present invention relates to a reaction analysis method including a procedure for dividing a liquid flowing through a flow path by introducing a fluid into the flow path and sending the liquid.

  In recent years, microchips having reaction regions and flow paths for performing chemical and biological analysis on a silicon or glass substrate have been developed by applying microfabrication technology in the semiconductor industry. These microchips are beginning to be used in, for example, electrochemical detectors for liquid chromatography and small electrochemical sensors in the medical field.

  Such an analysis system using a microchip is called μ-TAS (micro-total-analysis system), lab-on-chip, biochip, etc., and speeds up and increases the efficiency of chemical and biological analysis. As a technology that enables integration or downsizing of an analyzer, it has been attracting attention.

  μ-TAS can be used for biological analysis, especially for precious trace samples and many specimens, because it can be analyzed with a small amount of sample and disposable use of the chip is possible. Is expected.

  Patent Document 1 discloses a micropump system that performs pumping, mixing, and valve switching using bubbles generated in a microchannel (flow path) by a movable light beam, with respect to a liquid processing method in μ-TAS (the said (Refer to literature claims 1, 55, 56, and 60). This micropump system is intended for analysis such as diagnosis or high-throughput screening, or can be used for applications such as for the synthesis of combinatorial chemical libraries (see paragraph 0144).

  As an example of application of μ-TAS to biological analysis, the characteristics of microparticles such as cells are optically analyzed in a flow path provided on a microchip, and a population satisfying a predetermined condition from the microparticles. There is a fine particle sorting technology that separates and collects a group of particles (group).

  In relation to this fine particle sorting technique, Patent Document 2 discloses a particle sorting device using laser trapping. In this particle sorting apparatus, particles such as moving cells are irradiated with scanning light to apply an action force according to the type of particles to sort the particles.

  As a similar technique, Patent Document 3 discloses a particulate collection device that uses optical pressure or optical pressure. This fine particle collecting apparatus irradiates the flow path of fine particles with a laser beam so as to intersect the flow direction of fine particles, and deflects the moving direction of the fine particles to be collected in the convergence direction of the laser beam to collect the fine particles. .

  Patent Document 4 describes a microparticle sorting microchip having an electrode for controlling the moving direction of microparticles. This electrode is installed in the vicinity of the flow path from the fine particle measurement site to the fine particle sorting flow path, and controls the moving direction of the fine particles by interaction with the electric field.

JP-T-2005-538287 Japanese Patent Laid-Open No. 7-24309 JP 2004-167479 A JP 2003-107099 A

  As disclosed in Patent Documents 1 to 4, in the conventional μ-TAS, a predetermined chemical reaction is performed in the liquid continuously fed through the flow path.

  For this reason, a single reaction or a single continuous reaction is performed in the liquid flowing through the flow channel, and a plurality of independent chemical reactions cannot be performed in the flow channel.

The present invention provides a arranged flow paths in the substrate, and feeding and cutting the continuity of the liquid flowing through, by Rukoto the presence of a predetermined number of microparticles per shed liquid , the main purpose is to provide a technique for the chemical reaction in the one flow path to allow analysis.

  In order to solve the above problems, the present invention provides the following reaction analysis method.

  In other words, by introducing either a gas or an insulating liquid into the flow path disposed on the substrate, the liquid flowing through the flow path is divided and sent. A procedure for injecting a plurality of substances into the liquid, a procedure for injecting identification microbeads into the divided liquid, a procedure for detecting a reaction between the substances, and an identification signal from the identification microbeads. And a reaction analysis method including a detection procedure.

  In this reaction analysis method, the identification signal is detected by measuring at least one selected from temperature, fluorescence, scattered light, magnetization, charge, shape, or concentration of the identification microbead.

  Here, the term “liquid” in the present invention should be interpreted in a broad sense, and includes a homogeneous liquid, a suspension, that is, a liquid containing microparticles, a liquid containing small bubbles, an aqueous liquid, an organic liquid, It may include phase systems and hydrophobic and hydrophilic liquids.

  Also, the term “gas” should not be interpreted in a narrow sense, and can broadly include gases such as air and nitrogen.

  In the present invention, the term “microparticles” includes a wide range of microparticles such as cells, microorganisms, biological polymer materials such as liposomes, or synthetic particles such as latex particles, gel particles, and industrial particles. The cells of interest include animal cells (such as blood cells) and plant cells. Microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Biopolymer substances include chromosomes, liposomes, mitochondria, organelles (organelles) that constitute various cells. Furthermore, the microparticles | fine-particles which modified | denatured the surface or the inside of microparticles, such as glass and a polystyrene, chemically or physically modified | denatured bio-related high molecular substances, such as DNA, protein, and an antibody, may be sufficient. The industrial particles may be, for example, organic or inorganic polymer materials, metals, and the like. Organic polymer materials include polystyrene, styrene / divinylbenzene, polymethyl methacrylate, and the like. Inorganic polymer materials include glass, silica, magnetic materials, and the like. Metals include gold colloid, aluminum and the like. The shape of these fine particles is generally spherical, but may be non-spherical, and the size and mass are not particularly limited.

The present invention, in the arranged flow paths in the substrate, and feeding and cutting the continuity of the liquid flowing through, by Rukoto the presence of a predetermined number of microparticles per shed liquid one One technique for the chemical reaction to allow analysis within the channel is provided.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

1. FIG. 1 is a schematic diagram (top view) showing a flow path disposed on a substrate of a liquid delivery device according to a first embodiment of the present invention. A plurality of flow paths can be provided on the substrate, but here, one flow path is shown as a representative and described.

  In the figure, reference numeral 1 denotes a substrate, and reference numeral 11 denotes a flow path. It is assumed that the liquid (indicated by hatching in the drawing) introduced into the flow path 11 is sent in the direction of arrow F from the left to the right in the drawing.

  The substrate 1 is usually formed of glass or various plastics (PP, PC, COP, PDMS). In the case of glass, the substrate 1 is flown by wet etching or dry etching, and in the case of plastic by nanoimprinting, injection molding, or cutting. A path 11 is formed.

  As will be described later, it is preferable to form the substrate 1 using a water-repellent material in order to completely divide the liquid in the flow channel when the gas is introduced into the flow channel 11. Further, the surface of the flow path 11 may be subjected to water repellent processing. The water-repellent treatment is applied to the surface of the flow path in addition to the surface treatment by the application of normally used silicon resin-based water repellent and fluororesin-based water repellent, and film formation such as acrylic silicone water-repellent film and fluorine water-repellent film. Water repellency can also be imparted by forming a fine structure on the surface.

  In FIG. 1, reference numeral 12 denotes a fluid introduction part for introducing a fluid into the flow path 11. The fluid introduction part 12 communicates with the flow path 11 at one end, and introduces either a gas or an insulating liquid supplied from the other end by a delivery means (not shown) into the flow path 11. As the delivery means, a normally used pressure pump or the like can be adopted. Hereinafter, the part where the fluid introduction part 12 communicates with the flow path 11 is referred to as “connection part 13”, the upstream of the connection part 13 of the flow path 11 is referred to as “introduction path 111”, and the downstream of the connection part 13 is referred to as “liquid feed path 112”. Shall.

  The liquid feeding device according to the present invention introduces a fluid from the fluid introduction unit 12 to the connection unit 13 at a predetermined timing, so that the liquid fed from the introduction path 111 is divided by the fluid and sent to the liquid feeding path 112. Shed.

  Although FIG. 1 shows the case where one fluid introduction part 12 is provided, two or more fluid introduction parts 12 communicated in the connection part 13 can be provided. For example, the fluid introduction parts 12 and 12 may be provided on both sides of the connection part 13 (upper and lower sides in FIG. 1), and the fluid may be introduced into the connection part 13 from both directions of the flow path 11 (up and down direction in FIG. 1). .

2. FIG. 2 is a schematic diagram showing a flow path 11 through which a dispersion liquid of fine particles is passed in the liquid delivery apparatus according to the second embodiment of the present invention. FIG.

  In the figure, the symbol P indicates fine particles contained in the dispersion. Reference numeral 2 denotes a detection unit for detecting the fine particles P in the dispersion liquid fed through the introduction path 111.

  In the present embodiment, the detection unit 2 is disposed on both sides of the introduction path 111 on the substrate 1 as a set of microelectrodes, an AC voltage is applied between the electrodes, and a microscopic change is caused by a change in impedance flowing between the electrodes. A configuration for detecting the particles P is adopted.

  For example, the detection unit 2 may be configured to optically detect the microparticles P. For example, the detection unit 2 is configured by a known optical detection system in the microparticle sorting technique disclosed in Patent Documents 2 to 4 described above. be able to. In this case, the detection unit 2 is provided with a laser light source and an optical path for condensing and irradiating a predetermined portion of the introduction path 111 with the laser light from the light source. The microparticles P are detected by guiding and detecting light generated from the microparticles P in the introduction path 111 by laser light irradiation to the detector through the same or other optical paths. At this time, the light to be detected may be scattered light or fluorescence generated from the fine particles P by irradiation with laser light.

  When the detection unit 2 is configured as an optical detection system, the substrate 1 is preferably formed using a material that can transmit the laser light to be used and has a small wavelength dispersion with respect to the laser light and a small optical error.

  In the liquid feeding device according to the present invention, the connection portion 13 is controlled by controlling the opening / closing timing of a valve (not shown) of the delivery means of the fluid introduction portion 12 based on the detection signal of the fine particles P by the detection portion 2. In FIG. 2, a fluid is introduced between the fine particles P, and the dispersion is divided into a predetermined number of fine particles P and sent to the liquid feeding path 112 (FIG. 2 shows the dispersion divided into fine particles one by one. Shows the case).

  The number of fine particles P contained in the divided dispersion liquid can be arbitrarily set by controlling the opening / closing timing of the valve of the fluid introduction unit 12 according to the detection signal from the detection unit 2. .

  In FIG. 3, an example of the opening / closing timing of the valve | bulb of the fluid introduction part 12 is shown. In the upper part of the figure, the detection signals of the fine particles P by the detection unit 2 are shown in time series. In the lower part, an opening / closing signal input to the valve of the fluid introduction unit 12 is shown.

  FIG. 3 shows the opening / closing timing of the valve of the fluid introduction part 12 when the dispersion is divided for each fine particle P (see FIG. 2). As shown in the figure, for each detection signal of the microparticles P from the detection unit 2, an opening / closing signal is output to the valve of the fluid introduction unit 12 and the fluid is introduced into the connection unit 13, whereby the dispersion liquid is converted into the microparticles 1. It becomes possible to divide every one.

  At this time, the delay time of the opening / closing signal with respect to the detection signal is appropriately set according to the flow rate of the microparticles P in the introduction path 111 and the distance between the detection unit 2 and the connection unit 3.

  FIG. 4 shows another example of the opening / closing timing of the valve of the fluid introduction unit 12.

  FIG. 4 shows a case where an opening / closing signal is output to the valve of the fluid introduction unit 12 and a fluid is introduced into the connection unit 13 every time the detection signal of the microparticles P is output twice from the detection unit 2. In this case, as shown in FIG. 5, the dispersion liquid is divided into two fine particles and sent to the liquid feeding path 112.

  As shown in FIGS. 2 and 5, the fine particles P sent to the introduction path 111 do not necessarily need to be sent at equal intervals, and according to the detection signal of the fine particles P from the detection unit 2. By outputting an opening / closing signal to the valve of the fluid introduction unit 12, even when the microparticles P are sent at an indefinite interval, the dispersion liquid can be divided for each arbitrary number of microparticles P.

3. Next, the case where the fine particles P are sorted in the liquid flow feeding device according to the present invention will be described.

  FIG. 6 is a schematic diagram (top view) showing a flow path disposed on the substrate of the liquid delivery device according to the third embodiment of the present invention.

  The flow path 11 of the liquid feeding apparatus according to the present embodiment includes branch flow paths 113 and 114 that branch from the liquid feed path 112 at a branch portion indicated by reference numeral 14 in the drawing. In this liquid feeding apparatus, the dispersion liquid that is divided and fed in the liquid feeding path 112 can be selectively sent to one of the branch channel 113 and the branch channel 114.

  Control of the flow direction in the branching section 14 can be performed based on the charge or magnetization applied to the divided dispersion. The specific procedure and configuration will be described below.

  In FIG. 6, reference numeral 1111 denotes a charging unit for applying a voltage to the dispersion liquid in the introduction path 111. The charging unit 1111 applies a positive or negative voltage to the dispersion in the introduction path 111 when the fluid is introduced from the fluid introduction unit 12 to the connection unit 13. As a result, it is possible to impart a positive or negative charge to the dispersion that is divided into the liquid feeding path 112 by introduction of the fluid.

  The branch channels 113 and 114 are charged positively or negatively by a pair of positively or negatively charged electrodes 1131, 1131 and 1141, 1141 disposed on both sides thereof. The dispersion liquid charged by the charging unit 1111 and divided into the liquid feeding path 112 is sent to the branching channel charged in the branching unit 14 opposite to the charge.

  FIG. 6 shows a case where the electrodes 1131 and 1131 of the branch channel 113 are charged positively and the electrodes 1141 and 1141 of the branch channel 114 are negatively charged. As a result, a negatively charged dispersion liquid (indicated by hatching in the figure) can be sent to the branch flow path 113, and a dispersion liquid having positive charge (indicated by dots in the figure) can be sent to the branch flow path 114. This makes it possible to separate the fine particles P contained in the dispersion into two groups.

  In addition, when controlling the flow direction in the branching section 14 based on the magnetization applied to the dispersion, a substance capable of holding magnetization such as magnetic microbeads is mixed in the dispersion, and the substance and Based on the magnetic repulsive force with the magnetic field generated by a magnetic field generator such as a coil provided in the liquid feeding device, the branch flow path 113 or the branch flow path located on the N pole side or the S pole side in the branch portion 14 114 is sent.

  In order to maintain the charge or magnetization of the dispersion divided into the liquid feeding path 112, it is preferable to use a gas as the fluid. At this time, if the dispersion of the dispersion liquid in the liquid feed path 112 is incomplete and the adjacent dispersion liquid is partially connected, the charge and magnetization of the dispersion liquid are lost, and the flow direction is controlled. It may become impossible or inaccurate. Therefore, it is desirable to impart water repellency to the surface of the flow path 11 (liquid feeding path 112) in order to completely divide the dispersion with the introduced gas and maintain insulation between the divided dispersions. Furthermore, it is also effective to impart electric insulation to the surface of the flow path 11 (liquid feeding path 112) to prevent the movement of electric charges between the divided dispersions. The electrical insulation can be imparted, for example, by applying or forming a film having an insulation property on the channel surface. In addition, it is possible to prevent energization between the dispersions by flowing an insulating liquid such as ultrapure water on the flow path surface.

  In addition, in order to maintain the charge or magnetization of the dispersion liquid divided to the liquid feeding path 112, a liquid having electrical or magnetic insulation properties (“insulating liquid”) may be used as the fluid. For example, the above-described ultrapure water is used as the insulating liquid. Thereby, the movement of the electric charge or magnetization between the divided dispersion liquids can be prevented.

  The classification of the fine particles P can be performed based on the result of determining the characteristics of the fine particles P by the detection unit 2. In the present embodiment, the detection unit 2 is configured as the optical detection system described above, and the light generated by the irradiation of the laser light to the microparticles P in the introduction path 111 is detected, so that the detection of the microparticles P and the optics are performed. Judge the characteristics. The laser beam irradiation spot in the introduction path 111 is shown as a circular region surrounded by a dotted line in FIG.

  The parameters for analyzing the optical properties of the fine particles P include forward scattered light for measuring the size of the fine particles, side scattered light for measuring the structure, and Rayleigh scattering according to the target fine particles and the purpose of sorting. Or scattered light such as Mie scattering or fluorescence. The detection unit 2 analyzes the light detected by these parameters and determines whether or not the microparticles P have predetermined optical characteristics.

  When the detection unit 2 determines that the microparticles P have the desired optical characteristics, the detection unit 2 outputs a positive signal to the charging unit 1111, and the dispersion liquid in the introduction path 111 is either positive or negative. Charge. Here, the “positive signal” is a separation signal obtained from a dispersion liquid containing microparticles having desired characteristics, and the flow direction in the branching section 14 is controlled based on this separation signal (positive signal). Is carried out, and fine particles are collected.

  FIG. 7 shows an example of charging timing in the charging unit 1111. From the top in the figure, the detection signal from the detection unit 2 and the positive signal, the charging timing in the charging unit 1111, and the open / close signal input to the valve of the fluid introduction unit 12 are shown in time series.

  In the figure, for each detection signal of the microparticles P detected by the detection unit 2, an open / close signal is output to the valve, and a fluid is introduced into the connection unit 13 so that the dispersion liquid is divided for each microparticle P. When it is determined by the unit 2 that the microparticles P have desired optical characteristics and a positive signal is output, a case where positive charge is applied by the charging unit 1111 is shown. At this time, when it is determined by the detection unit 2 that the microparticles P do not have the desired optical characteristics and a positive signal is not output, the charging unit 1111 imparts negative charge.

  By controlling the charge in the charging unit 1111 at this timing, as shown in FIG. 6, only the fine particles P having desired optical characteristics are contained in the dispersion liquid having a positive charge, and the liquid supply path. It is possible to flow into the 112 and sort into the branch channel 114.

  The delay time, intensity, phase, pulse width, etc. of the charge and open / close signal with respect to the detection signal and positive signal are determined by the flow rate of the microparticles P in the introduction path 111 and the distance between the detection unit 2 and the connection unit 3. It is set accordingly.

  Moreover, when fractionation is performed based on the magnetization applied to the dispersion, the flow rate of the microparticles P in the introduction path 111 and the detection unit 2 and the branching unit 14 are detected with respect to the detection signal and the positive signal. Sorting can be performed by generating a magnetic field (or switching the magnetic field) at an appropriate timing in accordance with the distance and the like.

  FIG. 8 is a schematic diagram (top view) showing the flow path disposed on the substrate of the liquid delivery device according to the fourth embodiment of the present invention.

  In the present embodiment, the charging unit 1111 is a microelectrode disposed on the surface of the liquid feeding path 112, and a charge is imparted to the dispersion after being divided by introduction of fluid from the fluid introduction unit 12. Yes. In addition, the detection unit 2 is configured as a microelectrode, and the electrical characteristics of the microparticle P are determined, and the microparticle P can be sorted based on the result.

  The detection unit 2 detects changes in impedance flowing between the electrodes, detects the microparticles P, determines electrical characteristics, and outputs a signal as to whether or not the microparticles P are to be sorted. When a positive signal is output from the detection unit 2, the charging unit 1111, for example, adds a positive voltage to the dispersion liquid containing the microparticles P from the dispersion liquid that is divided and sent through the liquid supply path 112. Is applied, the fine particles P are sorted into the branch flow path 114.

  FIG. 9 shows an example of the charging timing in the charging unit 1111 in this embodiment. From the top in the figure, the detection signal and positive signal from the detection unit 2, the opening / closing signal for the valve of the fluid introduction unit 12, and the charging timing in the charging unit 1111 are shown in time series.

  As described above, according to the flow feeding method and the flow feeding device according to the present invention, the dispersion of fine particles flowing in the flow path is divided into a predetermined number of fine particles and dispersed in the divided dispersion. By applying an electric charge according to the characteristics of the microparticles contained in the liquid, it is possible to sort the microparticles.

  In this way, by separating the microparticles in the flow path based on the charge of the dispersion liquid, as in the past, a method of performing the sorting by applying an acting force directly to the microparticles and moving in the liquid, and Compared to the apparatus, the flow direction of the fine particles can be controlled quickly and easily, and the fine particles can be sorted at high speed and with high accuracy. In addition, it is possible to reduce the amount of liquid flowing through the flow path and collect the fine particles after sorting at a high concentration.

4). Next, a further embodiment of the liquid feeding method and the liquid liquid feeding apparatus according to the present invention will be described.

  FIG. 10 is a schematic diagram (top view) showing the flow path disposed on the substrate of the liquid delivery device according to the fifth embodiment of the present invention.

  In the present embodiment, in addition to the configuration described so far, an injection unit 3 for injecting a predetermined substance (see symbol S in the figure) into the dispersion liquid containing fine particles divided in the liquid supply path 112; A reaction detection unit 21 for detecting the reaction between the injected substance S and the fine particles P is provided.

  For example, when the microparticle P is a cell, a microorganism, or a biopolymer substance, the injection unit 3 is for injecting a physiologically active substance, an antibody, and various reagents capable of reacting with these into the dispersion. When the fine particles P are organic or inorganic polymer material or metal, for example, a compound capable of reacting with these is injected. Furthermore, the reaction detector 21 can inject an indicator capable of detecting the temperature or concentration, pH, etc. electrically or optically. As these substances, a plurality of substances may be injected simultaneously, or one or more substances may be selectively injected from a plurality of substances. In addition, a plurality of injection units 3 can be disposed in the liquid delivery unit 112, and a substance is injected into each divided dispersion liquid selectively from all the injection units or from any one of the injection units. You can also

  Similar to the detection unit 2, the reaction detection unit 21 is arranged on both sides of the liquid feeding path 111 as a set of microelectrodes, and an AC voltage is applied between the electrodes, and injection is performed by a change in impedance flowing between the electrodes. The reaction between the substance S injected from the part 3 and the fine particles P is detected. As described above, the detection unit 2 and the reaction detection unit 21 can also be configured as an optical detection system.

  Detection of the reaction between the substance S injected from the injection unit 3 and the microparticles P is detected by a change in the electrical characteristics or optical characteristics of the microparticles P caused by the reaction with the substance S. First, the detection unit 2 determines electrical characteristics or optical characteristics of the fine particles P that are sent through the introduction path 111. Next, after injecting the substance S from the injection unit 3 into the dispersion liquid divided in the liquid supply path 112, the reaction detection unit 21 determines the electric characteristic or the optical characteristic again. Then, the reaction between the substance S and the fine particles P is detected based on the change in the determination result of the electrical property or the optical property obtained by the detection unit 2 and the reaction detection unit 21.

  The reaction detection unit 21 outputs a positive signal when a reaction between the substance S and the fine particles P is detected. The charging unit 1111 gives positive or negative charge to the dispersion based on the positive signal. Thereby, the microparticles P in which the reaction has been detected can be sorted into one of the branch channel 113 or the branch channel 114 based on the charge of the dispersion.

  FIG. 11 shows an example of the charging timing in the charging unit 1111 in the present embodiment. From the top in the figure, the detection signal from the detection unit 2, the opening / closing signal input to the valve of the fluid introduction unit 12, the positive signal from the reaction detection unit 21, and the charging timing in the charging unit 1111 are shown in time series.

  As described above, according to the present embodiment, it is possible to sort out only the group that has reacted with the predetermined substance S among the fine particles P.

  As a specific example, if microbeads in which a nucleic acid probe is solid-phased are used as microparticles P and a nucleic acid chain containing a target nucleic acid chain and an intercalator fluorescent dye are injected as substance S, a nucleic acid probe and a target nucleic acid chain It is possible to detect only the hybridized microbeads by detecting the hybridization by fluorescence.

  Further, for example, when a cell is used as the microparticle P and a fluorescently labeled antibody against the cell surface antigen is used as the substance S, a specific surface antigen can be detected by selectively detecting fluorescence from the fluorescently labeled antibody bound to the cell surface antigen. It is possible to sort out only the cell group that expresses.

5. Reaction Analysis Method and Reaction Analysis Device FIG. 12 is a schematic diagram (flow path top view) for explaining a reaction analysis method using the liquid feeding method according to the present invention.

  In the figure, the reaction buffer sent from the introduction path 111 is divided by appropriately introducing a fluid from the fluid introduction section 12 and sent to the liquid sending path 112, and a predetermined reaction is performed in the divided reaction buffer. The method of performing detection is shown.

In FIG. 12, reference numerals 31 and 32 denote injection units for injecting a predetermined substance into the reaction buffer solution divided in the liquid supply path 112. In the figure, the case where the substance S ₁ is injected from the injection part 31 and the substance S ₂ is injected from the injection part 32 and the reaction between the substance S ₁ and the substance S ₂ is detected by the reaction detection part 21 is shown.

  A plurality of injection sections 31 and 32 can be arranged in the liquid supply path 112, and each of the divided reaction buffer solutions can be selectively supplied from all injection sections or from any one of the injection sections. An injection can be performed. In addition, a plurality of substances may be injected simultaneously from each injection part, or one or more substances may be selectively injected from the plurality of substances.

As an example, in order to screen a substance that can react with the single substance S ₁ injected from the injection unit 31, each of the reaction buffers obtained by dividing a plurality of candidate substances one by one as the substance S ₂ from the injection unit 32. The reaction is sequentially injected into the liquid, and the reaction detector 21 detects the presence or absence of a reaction in each reaction buffer. In addition, if a reaction buffer containing a predetermined substance in advance is introduced into the flow path 11, a candidate substance capable of undergoing a primary reaction with the substance is introduced from the injection unit 31 into a secondary reaction candidate capable of reacting with a primary reaction product. It is also possible to inject a substance from the injection part 32 and perform two-stage screening.

  Moreover, application to PCR is also possible. For example, a reaction solution containing a template DNA to be amplified in advance, a salt, nucleotides (dTNPs) and the like is introduced into the flow path 11, and forward and reverse primers are combined in a plurality of combinations from the injection part 31 and the injection part 32, respectively. By injecting and detecting the presence or absence of reaction in each reaction solution in the reaction detection unit 21, a primer set capable of efficiently amplifying the template DNA can be found.

As described with reference to FIG. 11, the reaction detection unit 21 is disposed on both sides of the liquid feeding path 111 as a set of microelectrodes, and an AC voltage is applied between the electrodes. detecting a reaction between the substance _{S 1} and the substance _{S 2.} Further, as described above, the optical detection system may be configured.

  In FIG. 12, reference numeral 4 denotes a bead introduction part for injecting identification microbeads (see reference numeral B in the figure) for identifying individual reaction buffers that have been divided.

  The identification microbeads B are magnetic beads or fluorescent beads having different electrical characteristics or optical characteristics, and can identify individual beads by obtaining characteristic values of electrical characteristics or optical characteristics as identification signals. Used. The identification signal of the identification microbead B can be detected by measuring its electrical characteristics or optical characteristics by the reaction detector 21. In addition, if fluorescent beads whose amount of fluorescence reversibly changes depending on temperature are used as the identification microbeads B, the reaction detection unit 21 can measure the temperature of each divided reaction buffer solution. . In addition, the identification microbeads B have a surface modification (processing) or internal that can be detected as an identification signal by measuring the temperature, fluorescence, scattered light, magnetization, charge, shape, concentration, etc. by the reaction detection unit 21. It is possible to employ various modified beads made of glass or polystyrene.

The bead introduction unit 4 introduces these beads into the divided reaction buffer before or after the injection of the substances S ₁ and S ₂ by the injection units 31 and 32.

For example, a single substance capable of reacting with the substance S ₁ to be injected from the injection unit 31 for the purpose of screening, from the injection unit 32 one by one a plurality of candidate substance as a substance S _2, each of the reactions were separated when sequentially injected into the buffer, the different identification micro beads B in accordance with the substance S ₂ which is injected from the injection unit 32 is introduced from the bead introduction part 4 in the reaction buffer. Then, the reaction detection unit 21 detects the reaction between the substance S ₁ and the substance S ₂ and detects the identification signal from the identification microbead B, whereby the correspondence relationship between the substance S ₂ and the identification microbead B is detected. Based on the above, it becomes possible to know which substance is the candidate substance injected into the reaction buffer in which the reaction is detected.

  As described above, based on the liquid feeding method according to the present invention, a reaction buffer solution flowing through the flow channel is divided, and various substances are injected into the divided reaction buffer solution, thereby providing one flow channel. It is possible to simultaneously analyze a plurality of chemical reactions. Therefore, the reaction analysis can be performed at a higher throughput than the conventional method in which the reaction is performed in the liquid continuously fed through the microchip channel.

  Note that, in the reaction analysis method based on the liquid feeding method according to the present invention, unlike the above-described separation of the fine particles, the fluid for dividing the reaction buffer solution may be a gas or a liquid, and this liquid May not necessarily have electrical or magnetic insulation. However, when the electrical or magnetic characteristic value of the identification microbead B is detected, it is preferable to use an insulating liquid.

It is a schematic diagram (top view) which shows the flow path arrange | positioned on the board | substrate of the liquid feeding apparatus which concerns on 1st embodiment of this invention. FIG. 6 is a schematic diagram (top view) showing a flow path 11 through which a dispersion liquid of fine particles is passed in a liquid delivery device according to a second embodiment of the present invention. It is a figure explaining an example of the opening-and-closing timing of the valve | bulb of the fluid introduction part 12 in 2nd embodiment. It is a figure explaining the other example of the opening-and-closing timing of the valve | bulb of the fluid introduction part 12 in 2nd embodiment. FIG. 5 is a schematic diagram (top view) showing a flow path 11 when a dispersion is divided into two fine particles in a liquid delivery device according to a second embodiment of the present invention. It is a schematic diagram (top view) which shows the flow path arrange | positioned on the board | substrate of the liquid feeding apparatus which concerns on 3rd embodiment of this invention. It is a figure explaining an example of the charge timing of the charge part 1111 in 3rd embodiment. It is a mimetic diagram (top view) showing a channel arranged on a substrate of a liquid sending device concerning a 4th embodiment of the present invention. It is a figure explaining an example of the charge timing of the charge part 1111 in 4th embodiment. It is a mimetic diagram (top view) showing a channel arranged on a substrate of a liquid sending device concerning a 5th embodiment of the present invention. It is a figure explaining an example of the charge timing of the charge part 1111 in 5th embodiment. It is a schematic diagram (flow path upper surface figure) explaining the reaction analysis method using the liquid feeding method which concerns on this invention.

DESCRIPTION OF SYMBOLS 1 Substrate 11 Flow path 111 Introduction path 112 Liquid supply path 113,114 Branch flow path 1111 Charge part 1131,1141 Electrode 12 Fluid introduction part 13 Connection part 14 Branch part 2 Detection part 21 Reaction detection part 3,31,32 Injection part 4 micro bead introduction part B identification beads P microparticles _{S, S 1, S 2} material

Claims (4)

By introducing either a gas or an insulating liquid into the flow path disposed on the substrate and controlling the delivery means of the fluid introduction unit based on the detection signal of the microparticles by the detection unit , A procedure for dividing a liquid dispersion of fine particles flowing through the flow path into a predetermined number of fine particles and feeding the liquid,
A procedure for optically detecting the reaction between the injected substance and the microparticles ;
And a charge procedure for applying a voltage to the divided dispersion . The reaction analysis method according to claim 1, wherein the flow path is provided with water repellency and electrical insulation on the surface of the flow path for introducing either the gas or the insulating liquid. Detecting the identification signal from the injected identification microbead,
The identification signal, the identification microbeads temperature, fluorescence, scattered light, magnetization, charge, claim 1 comprising the steps is detected by measuring one or more selected from any shape or concentration or 2 The reaction analysis method as described. By introducing either a gas or an insulating liquid into the channel disposed on the substrate and controlling the delivery means based on the detection signal of the microparticles of the optical detection system, the fluid passes through the channel. A fluid introduction part for dividing the dispersion of flowing microparticles into a predetermined number of microparticles;
An optical detection system for detecting microparticles and determining optical properties;
A charging unit for applying charge to the dispersion liquid divided based on the detection result;
A liquid delivery apparatus comprising: a branch channel and an electrode for separating fine particles by controlling a delivery direction of a dispersion liquid divided based on an applied charge.