Particulate dispensing device, microchip, and microchip module

The present invention relates to a microparticle sorter, a microchip and a microchip module. More specifically, after detecting the characteristics of the fine particles flowing through the flow path formed in the microchip inside the chip, the droplets containing the fine particles are ejected to the outside of the chip, and the droplets are based on the characteristics of the fine particles. The present invention relates to a fine particle sorting device or the like that controls the moving direction of the particles to sort.

Conventionally, in order to discriminate the characteristics of bio-related fine particles such as cells, microorganisms and liposomes, or synthetic particles such as latex particles, gel particles and industrial particles, a dispersion liquid of fine particles is introduced into the flow path. However, a device for optically measuring the characteristics of the fine particles introduced into the flow path is used.

In particular, for biological-related fine particles, a device called flow cytometry (flow cytometer) is widely used (see Non-Patent Document 1). Some flow cytometrys are intended only for measuring the characteristics of fine particles, and some are configured so that only fine particles having desired characteristics can be separated based on the measurement results. Of the latter, a device that specifically targets cells is called a "cell sorter". Currently, commercially available cell sorters can measure and sort the characteristics of cells at a high speed of several thousand to tens of thousands per second.

In conventional flow cytometry, characteristics such as the size and structure of fine particles such as cells and microbeads are measured as follows. First, a sample solution containing fine particles to be measured in the flow cell is flowed to the center of the laminar flow of the sheath liquid, and the fine particles are arranged in a row in the flow cell. Next, the optical detection unit irradiates the fine particles arranged in the flow cell and passing through with the measurement light, detects scattered light and fluorescence generated from the fine particles, and measures the characteristics of the fine particles. Subsequently, when the fine particles are separated, the sample liquid is discharged as droplets containing the fine particles into the space outside the flow cell, and the moving direction of the droplets is controlled to control the fine particles having desired characteristics. Divide the particles.

Patent Document 1 (Fig. 7) describes, as a conventional cell sorter, a fluid system for arranging cells stained with a fluorescent labeling reagent in a row in a flow cell, and scattered light by irradiating the cells with laser light. A device including an optical system for detecting fluorescence and a preparative system for controlling the moving direction of droplets ejected into a space outside the flow cell is disclosed.

In these conventional flow cytometrys (cell sorters), the flow cell parts that make up the flow path system are made of expensive quartz, and they are made up of orifice parts that are separate from this flow cell, so the user can easily dispose of them. Not a possible configuration. Therefore, even if the flow cell parts and the orifice parts are thoroughly cleaned each time the measurement is performed, there is a possibility that cross-contamination of the sample may occur between the measurements. Such cross-contamination between samples and the use of expensive flow cells and orifice parts are major obstacles, especially when stem cells or the like separated by a cell sorter are used for regenerative medicine.

As a technique for solving cross-contamination between samples and the use of expensive flow cells and orifice parts, in recent years, areas and channels for performing chemical and biological analysis have been provided on silicon or glass substrates. Microchips have been developed. Analysis systems using such microchips are called μ-TAS (micro-total-analysis system), lab-on-a-chip, biochip, and the like.

As an application example of μ-TAS to the microparticle sorting technology, the microparticle analysis technology that optically, electrically or magnetically analyzes the characteristics of the microparticles in the flow path or region arranged on the microchip is used. is there. For example, in Patent Document 2, a fine particle-containing solution introduction flow path, a sheath flow forming flow path arranged on at least one side of the flow path, and fine particles for measuring the introduced fine particles are described in Patent Document 2. A fine particle separation microchip having a measurement site and two or more fine particle separation flow paths for separating and collecting fine particles installed downstream of the fine particle measurement site is disclosed. This microchip has an electrode near the flow path port from the fine particle measurement site to the fine particle separation flow path. According to the microparticle sorting device provided with this microchip, it is possible to control the moving direction of the fine particles by the interaction with the electrode electric field to sort the fine particles.

In flow cytometry (cell sorter) that applies μ-TAS, the flow path system can be constructed with disposable microchips, so there is no cross-contamination of samples between measurements. Further, since the preparative system can be configured in the airtight flow path arranged on the chip, pollutants such as aerosols do not get mixed in the sample at the time of measurement. However, on the other hand, it is necessary to send a liquid containing fine particles at high pressure into the flow path arranged on the chip, and the fine particles are flowing through the liquid to control the movement direction of the fine particles. Need to be done in. Therefore, it is difficult to increase the flow speed and sorting speed of fine particles, and the characteristics of cells are measured and sorted at a high speed of several thousand to tens of thousands per second like conventional flow cytometry (cell sorter). It was difficult.

<p><patcit num="1"><text>JP-A-2007-46947</text></patcit><patcit num="2"><text>Japanese Unexamined Patent Publication No. 2003-107099</text></patcit></p>

<p><nplcit num="1"><text>"Cell Engineering Separate Volume Experimental Protocol Series Flow Cytometry Freedom", Hiromitsu Nakauchi, Shujunsha, 2nd Edition, August 31, 2006</text></nplcit></p>

<p> As described above, in the conventional flow cytometry (cell sorter), since the flow cells constituting the flow path system are not disposable, there is a possibility that cross contamination between samples may occur. Further, even in flow cytometry (cell sorter) to which μ-TAS is applied, there is a problem that it is difficult to increase the throughput of analysis because it is difficult to increase the flow speed and the preparative speed of fine particles.</p><p> Therefore, the present invention provides a fine particle sorting device capable of high-speed analysis, safe, high-speed, and inexpensive sorting by eliminating cross-contamination between samples and the use of expensive flow cells and orifice parts. The main purpose is to do.</p>

<p> In order to solve the above problems, in the present invention, (1) a sample flow path through which a liquid containing fine particles is passed and an orifice for discharging the liquid from the inside of the sample flow path to the space outside the chip are bonded to each other. A microchip formed by a sample flow path of an orifice part formed by a cavity of a microtube embedded between layers of a substrate, and (2) a vibrating element for ejecting a liquid into droplets at the orifice. 3) A charging means for applying an electric charge to the ejected droplets, and (4) irradiating fine particles flowing through the sample flow path upstream of the orifice in the liquid feeding direction to detect the light generated from the fine particles. Between the optical detection means to be used, (5) counter electrodes arranged to face each other with the moving particles sandwiched along the moving direction of the droplets ejected into the space outside the chip, and (6) counter electrodes. The cross-sectional shape of the flow path is sent to the sample flow path between the light irradiation part and the orifice part, which is provided with two or more recovery means for collecting the droplets that have passed through the light, and the light from the optical detection means is irradiated. Provided is a fine particle sorting device having a conversion flow path that changes from a quadrangular shape to a circular shape according to the liquid direction. Further, in the present invention, (1) a sample flow path through which a liquid containing fine particles is passed and an orifice in which the liquid is discharged from the inside of the sample flow path to the space outside the chip are formed by bonding substrate layers. (2) The sample flow path of the orifice part is composed of the lumen of the microtube embedded between the substrate layers, and (3) the predetermined part of the sample flow path irradiates the passing fine particles with light to form the fine particles. It is configured as a light irradiation part for detecting the generated light, and (3) the cross-sectional shape of the flow path changes from a square shape to a circular shape according to the liquid feeding direction in the sample flow path between the light irradiation part and the orifice part. Provided is a microchip in which a conversion flow path is configured.</p><p> In the microtubule sorting device and the microchip according to the present invention, it is preferable that the microtubule is made of metal or ceramic and a noble metal coating is formed on the surface of the lumen. Further, the cross-sectional area of the deformed flow path may be formed so as to gradually or gradually decrease according to the liquid feeding direction. The microchip may be formed with a suction flow path whose one end is communicated with the sample flow path. In this case, it is preferable that the fine particle sorting device is provided with a suction means that is connected to the other end of the suction flow path and applies a negative pressure to the flow path. Further, the microchip is provided with a microtubule for introducing a laminar flow of another liquid containing fine particles into the laminar flow of the liquid flowing through the sample flow path upstream of the light irradiation unit in the liquid feeding direction. You may. In this case, it is preferable that the communication port of the flow path connected to the suction means to the sample liquid flow path is provided downstream of the opening of the microtubule in the liquid feeding direction and upstream of the conversion flow path. It becomes. The microchip includes a holder for holding the microchip and a vibrating element arranged on the microchip for dropletizing and discharging the liquid at the orifice, and a liquid supply path to the sample flow path is connected to the microchip. A sheath port to be formed, a sample port to which a supply path of a liquid containing fine particles to the microtube is connected, and a suction port to connect the suction flow path to a negative pressure source are integrally arranged with a holder. Can be configured as a microchip module.</p><p> In the present invention, the "fine particles" broadly include biologically related fine particles such as cells, microorganisms and liposomes, or synthetic particles such as latex particles, gel particles and industrial particles.</p><p> Biologically-related microparticles include chromosomes, liposomes, mitochondria, organelles (organelles), etc. that make up various cells. Cells include animal cells (such as blood cell lineage cells) and plant cells. Microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Further, the bio-related microparticles can also include bio-related macromolecules such as nucleic acids, proteins, and complexes thereof. Further, the industrial particles may be, for example, an organic or inorganic polymer material, a metal, or the like. Organic polymer materials include polystyrene, styrene / divinylbenzene, polymethylmethacrylate and the like. Inorganic polymer materials include glass, silica, magnetic materials and the like. Metals include colloidal gold, 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.</p>

<p> INDUSTRIAL APPLICABILITY The present invention provides a fine particle sorting device capable of high-speed analysis, safe, high-speed and low-cost sorting by eliminating cross-contamination between samples and the use of expensive flow cells and orifice parts. ..</p>

<figref num="1">It is a figure explaining the schematic structure of the fine particle sorting apparatus A which concerns on this invention.</figref><figref num="2">It is a figure explaining the schematic structure of the fine particle sorting apparatus A which concerns on this invention.</figref><figref num="3">It is a figure explaining the schematic structure of the fine particle sorting apparatus A which concerns on this invention.</figref><figref num="4">It is a figure which shows typically the schematic structure of the fine particle sorting apparatus A.</figref><figref num="5">It is a figure explaining the modification of the microparticle sorting apparatus A which concerns on this invention.</figref><figref num="6">It is a figure which shows the schematic structure of the microchip 1 which concerns on this invention.</figref><figref num="7">It is sectional drawing explaining the structure of the sample flow path 11 in the vicinity of the microtubule 16 of the microchip 1 and the narrowing flow path 17, and the state of the sample liquid layer flow and the sheath liquid layer flow which flow.</figref><figref num="8">FIG. 5 is a schematic cross-sectional view illustrating the structure of the sample flow path 11 in the vicinity of the conversion flow path 13 and the orifice 12 of the microchip 1, and the state of the flowing sample liquid laminar flow and sheath liquid laminar flow.</figref><figref num="9">It is a figure which shows typically the structure of the conversion flow path 13 of the microchip 1 and the sample flow path 11 in the vicinity of the orifice 12, and the sample liquid and sheath liquid which are ejected as droplets from the orifice 12.</figref><figref num="10">FIG. 5 is a schematic cross-sectional view illustrating the structure of the sample flow path 11 in the vicinity of the conversion flow path 13 and the orifice 12 of the microchip 101, and the state of the flowing sample liquid laminar flow and sheath liquid laminar flow.</figref><figref num="11">It is a figure which shows typically the structure of the conversion flow path 13 of a microchip 101 and the sample flow path 11 in the vicinity of an orifice 12, and the sample liquid and sheath liquid which are made into droplets and discharged from an orifice 12.</figref><figref num="12">It is sectional drawing which explains the width and depth of a sample flow path 11. (A) shows the opening position of the microtubule 16, (B) shows the light irradiation unit 33, and (C) shows the cross section of the sample flow path 11 in the orifice 12.</figref><figref num="13">It is a figure explaining the structure of the microchip module which concerns on this invention.</figref><figref num="14">It is a figure which shows typically the sorting of the fine particles by the fine particle sorting apparatus A.</figref>

Hereinafter, suitable embodiments for carrying out the present invention will be described with reference to the drawings. In addition, the embodiment described below shows an example of a typical embodiment of the present invention, and the scope of the present invention is not narrowly interpreted by this. The explanation will be given in the following order. 1. Fine particle sorter 2. Microchip (1) First Embodiment (1-1) Sample flow path (1-2) Suction flow path (1-3) Microtubules and narrowing flow path (1-4) Light irradiation part (1-5) Conversion flow path and microtubules (2) Second embodiment (2-1) Conversion flow path and microtubules 3. Channel width and depth at each part of the microchip 4. Microchip module (1) Vibration element (2) Holder and port 5. Operation of micro flow separator

1. Fine particle sorter 1 to 3 are diagrams illustrating a schematic configuration of a fine particle sorting device according to the present invention. In the figure, the fine particle sorting device indicated by reference numeral A is the main body A.₁Cover A₂Further sorting cover A on the part protected by₃There is a microparticle collection area protected by. This fine particle collection site is the sorting cover A.₃Consists of a microchip 1 that is inserted and attached to the top opening of the. In Fig. 2, the block arrow indicates the sorting cover A of the microchip module whose component is the microchip 1.₃Indicates the insertion direction to. In FIG. 3, the sorting cover A is shown for convenience.₃The illustration of is omitted, and the sorting cover A is further omitted.₃Of the microchip modules inserted in, the parts other than the microchip 1 are omitted.

The microparticle collection area is the microchip 1 and the main body A.₁Includes an optical detection means 3 for irradiating a predetermined portion of the microchip 1 with light, a pair of counter electrodes 4, 4, and three recovery means (containers 51, 52, 53) provided in the above. Containers 51 to 53 are the main body A₁It is detachably attached to.

The configuration of the fine particle sorting field will be described in detail with reference to FIG. FIG. 4 is a diagram schematically showing a schematic configuration of the fine particle sorting device A. The figure shows the microchip 1, the optical detection means 3, the counter electrodes 4, 4, and the containers 51 to 53. In the figure, reference numeral 2 indicates a vibrating element arranged on the microchip 1. Reference numerals 6 and 6 indicate grounded electrodes that are grounded.

The microchip 1 is formed with a sample flow path 11 through which a liquid (sample liquid) containing fine particles to be sorted is passed. The optical detection means 3 irradiates a predetermined portion of the sample flow path 11 with light (measurement light), and detects light (measurement target light) generated from fine particles passing through the sample flow path 11. Hereinafter, the portion of the sample flow path 11 to which the measurement light from the photodetection means 3 is irradiated is referred to as a light irradiation unit.

The microchip 1 can be formed of glass or various plastics (PP, PC, COP, PDMS, etc.). It is desirable that the material of the microchip is a material that has transparency to the measurement light emitted from the optical detection means 3, has low autofluorescence, and has a small wavelength dispersion, and thus has a small optical error.

The sample flow path 11 to the microchip 1 can be formed by wet etching or dry etching of a glass substrate, or by nanoimprint, injection molding, or machining of a plastic substrate. The microchip 1 can be formed by sealing a substrate on which a sample flow path 11 or the like is formed with a substrate of the same material or a different material.

The optical detection means 3 can be configured in the same manner as the conventional flow cytometry. Specifically, an irradiation system consisting of a laser light source, a condensing lens that condenses and irradiates fine particles with laser light, a dichroic mirror, a bandpass filter, etc., and a measurement generated from the fine particles by irradiation with laser light. It is composed of a detection system that detects the target light. The detection system is composed of, for example, a PMT (photomultiplier tube), an area image sensor such as a CCD or CMOS element, and the like. In the figure, only the condenser lens is shown as the optical detection means 3. Further, in the figure, the case where the irradiation system and the detection system are configured by the same optical path is shown, but the irradiation system and the detection system may be configured by separate optical paths.

The measurement target light detected by the detection system of the optical detection means 3 is light generated from fine particles by irradiation of the measurement light, and is, for example, scattered forward scattered light, side scattered light, Rayleigh scattering, Mie scattering, or the like. It can be light or fluorescence. The light to be measured is converted into an electric signal, and the optical characteristics of the fine particles are detected based on this electric signal.

The sample liquid that has passed through the light irradiation unit is discharged into the space outside the chip from the orifice 12 provided at one end of the sample flow path 11. At this time, by vibrating the microchip 1 by the vibrating element 2, the sample liquid can be made into droplets and discharged into the space outside the chip. In the figure, reference numeral D indicates a droplet ejected into the space outside the chip.

The droplet D may contain fine particles to be sorted. The counter electrodes 4 and 4 are arranged along the moving direction of the droplets ejected into the space outside the chip, and are arranged so as to face each other with the moving droplets interposed therebetween. An electric charge is applied to the ejected droplet by a charging means (not shown), and the counter electrodes 4 and 4 determine the moving direction of the droplet by an electric repulsive force (or attractive force) with the electric charge applied to the droplet. Control and direct the droplets to any of the vessels 51-53. The containers 51 to 53 for collecting droplets may be a plastic test tube container or the like which is usually used as shown in the figure, or a dispensing plate having 96 wells or the like provided on a plastic substrate. It may be a container or the like.

In this way, the fine particle sorting device A performs up to the characteristic detection of the fine particles by the optical detection means 3 on the microchip 1, and then controls the moving direction of the fine particles in the space outside the chip. In the fine particle sorting device A, the movement direction of the droplet containing the fine particles is controlled by the counter electrodes 4 and 4 based on the optical characteristics of the fine particles detected by the optical detection means 3, which is desired. The fine particles having the characteristics can be collected in any of the containers 51 to 53 and sorted.

In the fine particle sorting device A, the optical detection means 3 may be replaced with, for example, an electrical or magnetic detection means. When detecting the characteristics of fine particles electrically or magnetically, microelectrodes are arranged on both sides of the sample flow path 11 so that they face each other, and the resistance value, capacitance value (capacitance value), inductance value, impedance, and electrode. The change value of the electric field between them, or the magnetization, magnetic field change, magnetic field change, etc. are measured. In this case, the fractionation of the fine particles is performed based on the electrical or magnetic properties of the fine particles.

In addition, here, the counter electrode 4 and the ground electrode 6 are attached to the main body A.₁Although the case where the electrodes are fixed to the side has been described as an example, these electrodes are used as the sorting cover A as shown in FIG.₃It may be provided on the side. That is, the counter electrode 4 is the sorting cover A.₃It is also possible to dispose of the counter electrode terminal 43 for electrically connecting the counter electrode 4 to the outside so as to be exposed on the outer surface on the inner surface of the cover of the base material constituting the above. Similarly, the ground electrode 6 also has the sorting cover A.₃The ground electrode terminal 63 for electrically connecting the ground electrode 6 to the outside may be arranged on the inner surface of the cover of the base material constituting the above so as to be exposed on the outer surface. The counter electrode terminal 43 and the ground electrode terminal 63 exposed on the outer side surface are the sorting cover A.₃Body A₁When attaching to the main body A₁It is electrically connected to the side.

In FIG. 5, reference numerals 511,521,531 are the sorting covers A.₃Indicates a distribution port for discharging droplets whose movement direction is electrically controlled by counter electrodes 4 and 4 into containers 51 to 53. Sorting cover A to prevent the counter electrode 4 and the ground electrode 6 from coming into contact with the droplets.₃As shown in the figure, it is preferable to provide a partition wall separating the moving space of the droplet and the counter electrode 4 or the ground electrode 6 on the base material constituting the above.

Hereinafter, the details of each configuration of the fine particle sorting device A and its functions will be described in order.

2. Microchip (1) First Embodiment (1-1) Sample flow path First, the microchip 1 according to the first embodiment will be described with reference to FIGS. 6 to 9. FIG. 6 is a diagram showing a schematic configuration of the microchip 1. The microchip 1 is formed with a sample inlet 15 into which the sample liquid is introduced, a sheath inlet 14 into which the sheath liquid is introduced, and a charged electrode inlet 20 into which a charged electrode (charging means) immersed in the sheath liquid is inserted. ing. The sheath liquid introduced into the sheath inlet 14 is passed through the charged electrode inlet 20, then branches in two directions, the positive direction and the negative direction of the Y axis, and is sent to the sample flow path 11 at approximately 90 degrees. After being bent once, it merges and the liquid is sent downstream.

(1-2) Suction flow path The microchip 1 is formed with a suction flow path 18 having one end communicated with the sample flow path 11. Reference numeral 181 indicates a communication port of the suction flow path 18 to the sample flow path 11. A suction outlet 19 to which a suction means (negative pressure source) (not shown) is connected is formed at an end of the suction flow path 18 opposite to the communication port 181. The suction means composed of a vacuum pump or the like applies a negative pressure to the suction flow path 18. When microparticles or bubbles are clogged in the sample flow path 11 (particularly, the conversion flow path 13 or the microtubule 121 described later), a negative pressure is applied to the suction flow path 18 by the suction means to apply a negative pressure to the sample flow path. The sample liquid and sheath liquid in 11 are sucked from the communication port 181. As a result, it is possible to temporarily reverse the flow of the sample liquid or the like in the sample flow path 11 and eliminate the clogging of fine particles and bubbles. As shown in the figure, the suction flow path 18 preferably has two flow paths arranged as a pair. By arranging two suction flow paths 18, even if one flow path is clogged with fine particles or bubbles that have flowed back from the sample flow path 11, the other flow path can function.

(1-3) Microtubules and narrowing flow path A microtubule 16 for introducing the sample liquid introduced from the sample inlet 15 into the sheath liquid laminar flow is arranged at a portion of the sample flow path 11 where the sheath liquid merges. The laminar flow of the sample liquid is introduced into the sheath liquid laminar flow that flows through the microtubule 16 and is introduced from the sheath inlet 14 and flows through the sample flow path 11. As a result, the sample liquid laminar flow can be sent downstream of the sample flow path 11 in a state of being surrounded by the sheath liquid laminar flow.

The communication port 181 of the suction flow path 18 to the sample liquid flow path 11 is preferably provided downstream of the opening 161 of the microtubule 16 in the liquid feeding direction. When the communication port 181 is provided upstream of the opening 161, a negative pressure is applied to the suction flow path 18 by the suction means, and when the sample liquid or the like in the sample flow path 11 is sucked and backflowed, the microtubules flow back. This is because there is a risk that air bubbles may enter the microtubule 16 through the opening 161 and become clogged.

In FIG. 6, reference numeral 17 indicates a narrowing flow path configured in the sample flow path 11. The narrowing flow path 17 is formed so that the area of the cross section perpendicular to the liquid feeding direction gradually or gradually decreases from the upstream to the downstream in the liquid feeding direction.

FIG. 7 is a schematic cross-sectional view illustrating the structure of the sample flow path 11 in the vicinity of the arrangement portion of the microtubule 16 and the narrowing flow path 17, and the state of the flowing sample liquid laminar flow and sheath liquid laminar flow. .. (A) shows a horizontal sectional view (XY sectional view), and (B) shows a vertical sectional view (ZX sectional view). In the figure, reference numeral S indicates a sample laminar flow, reference numeral T indicates a sheath laminar flow, and reference numeral P indicates fine particles contained in the sample solution. Reference numerals 1a and 1b indicate a substrate layer. The flow path such as the microchip 1 and the sample flow path 11 and the orifice 12 are formed by laminating these substrate layers.

The sample laminar flow S is introduced into the sheath laminar flow T flowing through the sample flow path 11 by microtubules 16, and is surrounded by the sheath laminar flow T (three-dimensional laminar flow) as shown in the figure. ) And the liquid is sent.

The flow path side wall of the narrowing flow path 17 is formed so as to narrow in the Y-axis direction in the drawing according to the liquid feeding direction, and the narrowing flow path 17 has a pyramid shape that gradually becomes thinner in the top view thereof. Due to this shape, the narrowing flow path 17 narrows the laminar flow widths of the sheath liquid and the sample liquid in the Y-axis direction in the drawing and sends the liquid. Further, the narrowing flow path 17 is formed so that the bottom surface of the flow path becomes an inclined surface that becomes higher in the depth direction (Z-axis positive direction) from the upstream to the downstream, and the laminar flow width is also in the same direction. To narrow down.

In this way, the sample laminar flow S forms a three-dimensional laminar flow surrounded by the sheath laminar flow T, and the laminar flow width of the three-dimensional laminar flow is narrowed down to feed the sample liquid. Fine particles P can be arranged one by one in the laminar flow S and sent. Then, the flow position of the fine particles P in the sample flow path 11 can be positioned, and the measurement light from the optical detection means 3 can be accurately irradiated to the fine particles P.

In particular, according to the narrowing flow path 17, the sample liquid flow S flows not only in the horizontal direction of the microchip 1 (FIG. 7 (A) Y-axis direction) but also in the vertical direction (FIG. 7 (B) Z-axis direction). Since the laminar flow width can be narrowed down, the focal position of the measurement light in the depth direction of the sample flow path 11 can be precisely matched with the flow position of the fine particles P. Therefore, it is possible to obtain high measurement sensitivity by irradiating the fine particles P with measurement light with high accuracy.

Here, the sample flow path 11 is formed as a sufficiently thin flow path, and the sample liquid layer flow S is introduced into the sheath liquid laminar flow T passing through the sample flow path 11 by using a microtubule 16 having a small diameter. For example, it is possible to form a three-dimensional laminar flow in which the laminar flow width is narrowed down in advance. However, in this case, by reducing the diameter of the microtubule 16, the microtubule P may be clogged with the microtubules P.

In the microchip 1, by providing the narrowing flow path 17, a three-dimensional laminar flow was formed using a microtubule 16 having a diameter sufficiently larger than the diameter of the fine particles P contained in the sample liquid. Later, the laminar flow width can be narrowed down. Therefore, the problem of clogging of the microtubule 16 as described above does not occur.

FIG. 7 shows a case where the microtubule 16 is arranged so that its center is located coaxially with the center of the sample flow path 11. In this case, the sample laminar flow S is introduced at the center of the sheath laminar flow T passing through the sample flow path 11. The position of the sample laminar flow S in the sheath laminar flow T can be arbitrarily set by adjusting the opening position of the microtubule 16 in the sample flow path 11. Further, in order to narrow down the laminar flow width, the narrowing flow path 17 may be formed so that the area of the cross section perpendicular to the liquid feeding direction gradually decreases from the upstream to the downstream of the flow path, as shown in the figure. The shape is not limited to the above shape, and for example, both the bottom surface and the top surface of the flow path can be formed as inclined surfaces to narrow down the flow.

The inner diameter of the microtubule 16 can be appropriately set according to the diameter of the microtubules P to be sorted. For example, when blood is used as a sample solution and blood cell cells are analyzed, the inner diameter of a suitable microtubule 16 is about 10 to 500 μm. Further, the width and depth of the sample flow path 11 at the opening position of the microtubule 16 may be appropriately set according to the outer diameter of the microtubule 16 reflecting the diameter of the microtubule P. For example, when the inner diameter of the microtubule 16 is about 10 to 500 μm, the width and depth of the sample flow path 11 at the opening position of the microtubule 16 are preferably about 100 to 2000 μm, respectively. The cross-sectional shape of the microtubule can be any shape such as an ellipse, a quadrangle, or a triangle, in addition to the circular shape.

The laminar flow width of the sample laminar flow S and the sheath laminar flow T before narrowing down in the narrowing flow path 17 may change depending on the width and depth of the sample flow path 11 and the diameter of the microtubule 16, but the narrowing down. The area of the cross section perpendicular to the liquid feeding direction of the inlet 17 can be narrowed down to an arbitrary laminar flow width by appropriately adjusting the area. For example, in FIG. 7B, the flow path length of the narrowing flow path 17 is L, and the inclination angle of the bottom surface of the flow path is θ.₃If, the narrowing width of the three-dimensional laminar flow in the narrowing flow path 17 is L · tan θ.₃Will be. Therefore, the flow path length L and the inclination angle θ₃It is possible to set an arbitrary narrowing width by appropriately adjusting. Furthermore, in FIG. 7 (A), the stenosis angles of the narrowing flow path 17 flow path side walls in the Y-axis direction are θ.₁, Θ₂And these and the above θ₃To "θ₃= 2 × θ₁, Θ₁= θ₂By forming so as to be, the sample laminar flow S and the sheath laminar flow T are isotropically reduced, and the laminar flow width is narrowed without disturbing the three-dimensional laminar flow formed by the microtubules 16. be able to.

Here, in the microchip according to the present invention, the narrowing flow path 17 is not an essential configuration. For example, the sample flow path 11 is formed as a sufficiently thin flow path, and the sample liquid layer flow S is introduced into the sheath liquid laminar flow T passing through the sample flow path 11 by using a microtubule 16 having a small diameter. If a three-dimensional laminar flow in which the laminar flow width is narrowed in advance can be formed, the narrowing flow path 17 may not be provided. That is, the flow path width and depth of the microtubule 16 arrangement portion and the light irradiation portion described below may be the same. Further, as the microchip according to the present invention, it is not excluded that the flow path width and depth of the light irradiation portion are larger than the flow path width and the like of the arrangement portion of the microtubule 16.

(1-4) Light irradiation part In FIG. 6, reference numeral 33 indicates a light irradiation unit to which the measurement light from the optical detection means 3 is irradiated. The light irradiation unit 33 detects the measurement target light generated from the fine particles by irradiating the measurement light from the optical detection means 3.

As described above, in the light irradiation unit 33, since the laminar flow widths of the sample laminar flow and the sheath laminar flow are narrowed down by the narrowing flow path 17, the sample liquid laminar flow S in the sample flow path 11 It is possible to precisely match the focal position of the measurement light with the flow position and accurately irradiate the fine particles with the measurement light.

The laminar flow width of the sample laminar flow S and the sheath laminar flow T in the light irradiation unit 33 shall be an arbitrary laminar flow width by appropriately adjusting the area of the cross section perpendicular to the liquid feeding direction of the narrowing flow path 17. However, the width and depth of the sample flow path 11 are preferably about 20 to 2000 μm, respectively.

(1-5) Conversion flow path and microtubules In FIG. 6, reference numeral 12 indicates an orifice that discharges the sheath liquid and the sample liquid that have passed through the light irradiation unit 33 into the space outside the chip. The sheath liquid and the sample liquid are dropletized at the orifice 12 by the action of the vibrating element 2 described below, and are discharged to the outside of the chip.

The orifice 12 is formed by the bonded substrate layers 1a and 1b, but when the orifice 12 is formed only by bonding the substrate layers, the following problems occur. That is, first, when forming an orifice in each of the substrate layers 1a and 1b in a semicircular shape, high accuracy is required for forming the mold, and the diameter and roundness of both semicircular shapes have an error of several to several tens of μm. It is difficult to match with. Further, when bonding the substrate layer 1a, a 1b, it is required high accuracy in alignment of both semi-circular, Oh high roundness it is difficult to create the orifice. If the roundness of the orifice is low, the shape of the ejected droplets becomes irregular, and the control accuracy of the moving direction by the counter electrodes 4 and 4 deteriorates. Further, when changing the diameter of the orifice, it is necessary to recreate the mold, which causes a problem of high cost.

Therefore, in the microchip 1, the sample flow path 11 of the orifice portion is composed of the lumen of the microtubule 121 embedded between the substrate layers 1a and 1b. In the microtubule 121, the microtubule 121 is embedded in a groove formed coaxially with the sample flow path 11, sealed with an adhesive, and the sample liquid or the like sent to the sample flow path 11 is introduced into the cavity. Arranged to be. The sample liquid or the like introduced into the lumen of the microtubule 121 is discharged from the orifice 12 that coincides with the end of the microtubule 121.

By configuring the orifice part with microtubules 121 in this way, an orifice with high roundness can be created, the shape of the ejected droplets is stable, and the control of the moving direction by the counter electrodes 4 and 4 is reproducible. It is possible to perform with high accuracy. Further, since only the groove for embedding the microtubule 121 needs to be formed in the substrate layers 1a and 1b, the allowable error range in the alignment at the time of mold making and bonding can be increased, and the chip manufacturing cost can be reduced. Is possible. Furthermore, the diameter of the orifice can be easily changed by appropriately changing the inner diameter of the microtubule 121, and it is also necessary to recreate the mold by changing only the inner diameter while keeping the outer diameter of the microtubule 121 as it is. It is low cost because there is no such thing.

Here, the case where the sample flow path 11 and the groove for embedding the microtubule 121 are formed in the substrate layer 1b and bonded to the substrate layer 1a has been described as an example, but the sample flow path 11 and the groove are described in the substrate layer 1a. A part of each of 1b and 1b may be molded and bonded together.

The microtubule 121 can be made of metal or ceramic, quartz, or resin, and is preferably made of metal or ceramic. It is preferable to form a noble metal coating such as gold or platinum on the luminal surface of the microtubule 121. By using metal or ceramic, the durability of the orifice can be improved. Further, by forming a noble metal film on the surface of the lumen, it is possible to prevent the fine particles from adhering to the surface of the lumen or clogging the lumen, particularly when the fine particles are made into cells or the like. By setting the length of the flow path of the orifice portion formed of the microtubule 121 to 3000 m or less, preferably 100 to 500 μm or less, and more preferably 100 to 300 μm or less, the loss of liquid feeding pressure can be suppressed.

Reference numeral 13 indicates a conversion flow path configured in the sample flow path 11 upstream of the orifice 12 in the liquid feeding direction and downstream of the light irradiation unit 33. The conversion flow path 13 is a flow path for shifting the cross-sectional shape of the sample flow path 11 to the cross-sectional shape of the microtubule 121. That is, the conversion flow path 13 is configured so that the cross-sectional shape of the flow path changes from a quadrangular shape to a circular shape according to the liquid feeding direction (see also FIG. 9).

Further, the conversion flow path 13 is formed so that the area of the cross section perpendicular to the liquid feeding direction gradually or gradually decreases according to the liquid feeding direction. That is, similarly to the narrowing flow path 17, the side wall of the flow path is formed so as to narrow in the Y-axis direction in the figure according to the liquid feeding direction, and the bottom surface of the flow path is formed in the depth direction (Z-axis) from the upstream to the downstream. It is formed so as to be an inclined surface that rises in the positive direction).

FIG. 8 is a schematic cross-sectional view illustrating the structure of the sample flow path 11 in the vicinity of the conversion flow path 13 and the orifice 12, and the state of the flowing sample liquid laminar flow and sheath liquid laminar flow. (A) shows a horizontal sectional view (XY sectional view), and (B) shows a vertical sectional view (ZX sectional view). In the figure, reference numeral S indicates a sample laminar flow, reference numeral T indicates a sheath laminar flow, and reference numeral P indicates fine particles contained in the sample solution. Further, FIG. 9 is a diagram schematically showing the structure of the conversion flow path 13 and the sample flow path 11 in the vicinity of the orifice 12, and the sample liquid and the sheath liquid which are dropletized and discharged from the orifice 12.

The conversion flow path 13 is formed so that the cross-sectional shape of the flow path changes from a quadrangular shape to a circular shape according to the liquid feeding direction, and the cross-sectional area becomes smaller. As a result, the sample laminar flow S and the sheath laminar flow T are narrowed down in the Y-axis direction and the Z-axis direction in the figure while maintaining the three-dimensional laminar flow formed by the microtubules 16. It is introduced into the lumen of microtubule 121. By narrowing the laminar flow width, the conversion flow path 13 increases the feed pressure of the sample liquid and the sheath liquid in the sample flow path 11, and discharges them from the orifice 12 at a high pressure. By increasing the discharge pressure of the sample liquid or the like from the orifice 12, droplets can be formed at a high frequency at the orifice 12, and fine particles can be separated at high speed. In the figure, the moving direction of the ejected droplets is indicated by reference numeral F.

Since the laminar flow width is largely narrowed in the conversion flow path 13 and the lumen of the microtubule 121, there is a possibility that microparticles and bubbles may be clogged. When the fine particles are clogged, a negative pressure is applied to the suction flow path 18 by the above-mentioned suction means to temporarily reverse the flow of the sample liquid or the like to clear the clogging of the fine particles. Therefore, the communication port 181 of the suction flow path 18 to the sample flow path 11 is provided upstream of the conversion flow path 13 in the liquid feeding direction.

The laminar flow widths of the sample laminar flow S and the sheath laminar flow T in the microtubule 121 can be narrowed down to an arbitrary laminar flow width by appropriately adjusting the area of the cross section perpendicular to the liquid feeding direction of the conversion flow path 13. it can. For example, in FIG. 8B, the flow path length of the conversion flow path 13 is l, and the inclination angle of the bottom surface of the flow path is θ.₃If, the narrowing width of the three-dimensional laminar flow in the conversion flow path 13 is L · tan θ.₃Will be. Therefore, the flow path length l and the inclination angle θ₃It is possible to set an arbitrary narrowing width by appropriately adjusting. The laminar flow width (diameter) of the sample laminar flow S and the sheath laminar flow T in the microtubule 121 is preferably about 20 to 500 μm.

The laminar flow widths of the sample laminar flow S and the sheath laminar flow T can be narrowed down by using both the bottom surface and the top surface of the conversion flow path 13 as inclined surfaces, and the shape of the conversion flow path 13 is shown in the figure. The point that is not limited to the shape shown in is the same as that of the narrowing flow path 17. Further, in FIG. 8 (A), the stenosis angle θ in the Y-axis direction of the side wall of the conversion flow path 13 flow path₁, Θ₂And the stenosis angle θ in the Z-axis direction₃, "Θ₃= 2 × θ₁, Θ₁= θ₂The narrowing flow path 17 also explains that the three-dimensional laminar flow formed by the microtubules 16 can be isotropically reduced and the laminar flow width can be narrowed without being disturbed. That's right.

Here, in the microchip according to the present invention, the area of the cross section of the conversion flow path 13 perpendicular to the liquid feeding direction may not be formed so as to become smaller according to the liquid feeding direction. For example, when the laminar flow width of the three-dimensional laminar flow is sufficiently narrowed by the narrowing flow path 17 described above, the cross section of the conversion flow path 13 may be changed only in shape. Further, for example, even when the inner diameter of the microtubule 121 is sufficiently large with respect to the flow path width and depth of the light irradiation portion, the cross section of the conversion flow path 13 may be changed only in shape. That is, in these cases, the cross-sectional area of the flow path of the light irradiation unit and the cross-sectional area of the microtubule 121 may be the same. Further, depending on the inner diameter of the microtubule 121 used, it is not excluded that the microchip according to the present invention has a cross-sectional area of the conversion flow path 13 larger than the cross-sectional area of the flow path of the light irradiation portion. To do.

(2) Second embodiment (2-1) Conversion flow path and microtubules Next, the microchip 101 according to the second embodiment will be described with reference to FIGS. 10 and 11.

The configuration of the microchip 101 is the same as that of the microchip 1 according to the first embodiment with respect to the sample flow path, the suction flow path, the micro tube, the narrowing flow path, and the light irradiation unit, except for the conversion flow path and the micro tube. Is. Therefore, only the configuration of the conversion flow path and the microtubule of the microchip 101 will be described below.

FIG. 10 is a schematic cross-sectional view illustrating the structure of the sample flow path 11 in the vicinity of the conversion flow path 13 and the orifice 12, and the state of the flowing sample liquid laminar flow and sheath liquid laminar flow. (A) shows a horizontal sectional view (XY sectional view), and (B) shows a vertical sectional view (ZX sectional view). In the figure, reference numeral S indicates a sample laminar flow, reference numeral T indicates a sheath laminar flow, and reference numeral P indicates fine particles contained in the sample solution. Further, FIG. 11 is a diagram schematically showing the structure of the conversion flow path 13 and the sample flow path 11 in the vicinity of the orifice 12, and the sample liquid and the sheath liquid which are dropletized and discharged from the orifice 12.

The microchip 101 is different from the above-mentioned microchip 1 in that, of the sample flow paths 11, the conversion flow path 13 is also composed of the lumen of the microtubule 121 in addition to the flow path of the orifice portion.

The microtubule 121 is embedded in a hole formed coaxially with the sample flow path 11 between the substrate layers 1a and 1b, sealed with an adhesive, and the sample liquid or the like sent to the sample flow path 11 is discharged. It is arranged to be introduced into the lumen. On the outer peripheral surface of the microtubule 121, notches and convex portions for enhancing the adhesiveness to the substrate layers 1a and 1b are provided in a circumferential shape.

The conversion flow path 13 is formed so that the cross-sectional area of the sample flow path is greatly expanded at the end face of the microtubule 121 and then becomes smaller in the liquid feeding direction. By expanding the cross-sectional area of the flow path at the inlet of the microtubule 121 of the sample flow path in this way, the sample liquid laminar flow S and the sheath liquid laminar flow T are maintained on the Y-axis in the figure while maintaining the three-dimensional laminar flow. The laminar flow width can be narrowed down in the direction and the Z-axis direction. The conversion flow path 13 increases the feed pressure of the sample liquid and the sheath liquid in the sample flow path 11 by narrowing the laminar flow width, and discharges the sample liquid and the sheath liquid from the orifice 12 at a high pressure. By increasing the discharge pressure of the sample liquid or the like from the orifice 12, droplets can be formed at the orifice 12 at a high frequency, and fine particles can be separated at high speed. In the figure, the moving direction of the ejected droplets is indicated by reference numeral F.

In the figure, the conversion flow path 13 is shown so that the cross-sectional shape of the flow path changes from a quadrangular shape to a circular shape according to the liquid feeding direction. However, in the microchip according to the present embodiment, the cross-sectional shape of the conversion flow path 13 is changed. It may be consistently circular. That is, when the cross section of the flow path at the inlet of the microtubule 121 of the sample flow path is sufficiently expanded as compared with the cross section of the flow path at the orifice portion, the conversion flow path 13 can have a conical shape.

3. Channel width and depth at each part of the microchip FIG. 12 is a schematic cross-sectional view illustrating the width and depth of each portion of the sample flow path 11. The figure shows the YZ cross section of the sample flow path 11, where (A) shows the opening position of the microtubule 16, (B) shows the light irradiation unit 33, and (C) shows the cross section of the sample flow path 11 in the orifice 12.

As shown in FIG. 12 (A), at the opening position of the microtubule 16, the sample laminar flow S and the sheath laminar flow T are three-dimensional layers in which the sheath laminar flow T surrounds the sample laminar flow S. It is sent as a flow. As described above, the width and depth of the sample flow path 11 at the opening position of the microtubule 16 are appropriately set according to the outer diameter of the microtubule 16 reflecting the diameter of the microtubule P, for example, 100 to 2000 μm. It is said to be a degree.

The three-dimensional laminar flow formed by the microtubules 16 is sent to the light irradiation unit 33 in a state where the laminar flow width is narrowed by the narrowing flow path 17 (see FIG. 7). By narrowing the laminar flow width by the narrowing flow path 17, fine particles P are arranged one by one in the sample liquid laminar flow S and sent to the light irradiation unit 33.

The laminar flow widths of the sample laminar flow S and the sheath laminar flow T in the light irradiation unit 33 can be arbitrarily set by appropriately adjusting the area of the cross section perpendicular to the liquid feeding direction of the narrowing flow path 17. The width (W) and depth (H) of the sample flow path 11 in the light irradiation unit 33 are set to about 20 to 2000 μm, respectively, in order to sufficiently increase the optical detection angle (numerical aperture of the optical system) by the photodetecting means 3. It is preferable to do so.

Further, the shape of the sample flow path 11 in the light irradiation unit 33 may be a rectangular shape with respect to the irradiation direction of the light measured by the light detection means 3 by increasing the width (W) with respect to the depth (H). preferable. By forming the sample flow path 11 in the light irradiation unit 33 into such a wide shape, it is possible to increase the numerical aperture of the optical system.

The sample laminar flow S and the sheath laminar flow T that have passed through the light irradiation unit 33 are narrowed down again by the conversion flow path 13 as shown in FIG. 8 and sent to the orifice 12. By narrowing the laminar flow width by the conversion flow path 13, the discharge pressure of the sample liquid and the sheath liquid from the orifice 12 can be increased.

The laminar flow widths of the sample laminar flow S and the sheath laminar flow T at the orifice 12 can be arbitrarily set by appropriately adjusting the area of the cross section perpendicular to the liquid feeding direction of the conversion flow path 13. In order to form high-speed, high-frequency droplets at the orifice 12, the laminar flow widths of the sample laminar flow S and the sheath laminar flow T at the orifice 12 are reduced to sufficiently discharge the sample liquid and the sheath liquid. It is preferable to increase it to. Therefore, it is preferable that the inner diameter d of the microtubule 121 constituting the flow path of the orifice portion is about 20 to 500 μm.

4. Microchip module (1) Vibration element FIG. 13 is a diagram showing a configuration of a microchip module including the microchip 1 as an element.

In the figure, reference numeral 2 indicates a vibrating element arranged on the microchip 1. The vibrating element 2 vibrates the microchip 1 to atomize the sample liquid and the sheath liquid at the orifice 12 and discharge them into the space outside the chip. Further, the vibrating element 2 sets the vibration of the microchip 1 to a predetermined frequency to dropletize the sample liquid or the like so that the ejected droplet D contains the fine particles P one by one (also in FIG. 6). reference).

At this time, the vibration frequency of the vibrating element 2 is the flow velocity (flow velocity) of the fine particles P detected by the optical detection means 3 in the light irradiation unit 33 (see FIG. 3), the resonance frequency of the microchip 1, and the orifice 12. It is set according to the liquid feed pressure, the diameter of the orifice 12, and the like.

Droplets of the sample liquid and the sheath liquid using such a vibrating element can be performed in the same manner as flow cytometry using a conventional flow cell. As the vibrating element 2, for example, a piezo vibrating element or the like which is also used in an inkjet printer is used.

The vibrating element 2 covers the back surface of the microchip 1, that is, the microchip module with the sorting cover A.₃Main body A in the state of being inserted and attached to₁It is preferably placed on the side surface (see also Figure 4). By arranging the vibrating element 2 on the back surface, the sample flow path is not covered by the vibrating element 2 when the microchip module is attached. Therefore, it is possible to secure the visibility of the sample flow path and confirm whether or not the flow path is clogged with fine particles or bubbles. Further, the vibrating element 2 is preferably arranged at a position close to the orifice 12 in order to efficiently transmit the vibration to the orifice 12. The vibrating element 2 is the main body A.₁It may be arranged on the side, and in this case, when the microchip module is attached (see FIG. 3), the main body A is in contact with a part of the microchip 1.₁It may be arranged in.

(2) Holder and port In FIG. 13, reference numeral 7 indicates a holder that holds the microchip 1 and functions as an adapter for attaching the microchip 1 to the device body. It is preferable that the holder 7 is made of a material having the same light transmittance as that of the microchip 1 and ensures visibility of the sample flow path, the suction flow path, and the like formed on the microchip 1. As a result, when the flow path is clogged with fine particles or bubbles, the clogged portion can be easily confirmed.

A suction port 71, a sheath port 72, a sample port 73, and a connector 74 are arranged in a straight line on the holder 7. The suction port 71 communicates with the suction outlet 19 to which a negative pressure source is connected. The sheath port 72 and the sample port 73 communicate with the sample inlet 15 and the sheath inlet 14, respectively, and the sample liquid or the sheath liquid supply path is connected.

The connector 74 is formed by integrating two electrodes for the vibrating element 2 and one charged electrode, and wiring is connected to these electrodes from the main body. Wiring to the vibrating element 2 arranged on the back surface of the microchip 1 extends from the electrode for the vibrating element 2 of the connector 74. Further, the charged electrode of the connector 74 is inserted into the charged electrode inlet 20 of the microchip 1 and immersed in the sheath liquid. The charged electrode functions as a charging means for imparting a positive or negative charge to the sheath liquid and the sample liquid flowing through the sample flow path 11. The sample liquid and the sheath liquid are dropletized at the orifice 12 provided at one end of the sample flow path 11 and discharged into the space outside the chip. At this time, by applying a voltage to the charged electrode, a positive or negative charge can be applied to the ejected droplets.

In the microchip module according to the present invention, the suction outlet 19, the sample inlet 15, the sheath inlet 14, and the charged electrode inlet 20 are arranged in a row at the center of the microchip (center in the Y-axis direction in the figure), and the corresponding suction is performed. The port 71, the sheath port 72, the sample port 73, and the connector 74 are linearly arranged on the holder 7. This enhances the visibility of the sample flow path, the suction flow path, and the like formed on the microchip 1.

5. Operation of fine particle sorter Subsequently, the operation of the fine particle sorting device A will be described with reference to FIG.

The sample liquid and the sheath liquid that have passed through the light irradiation portion of the sample flow path 11 are discharged from the orifice 12 into the space outside the chip. In the light irradiation unit, the optical detection means detects the optical characteristics of the fine particles, and at the same time, detects the flow velocity (flow velocity) of the fine particles, the interval between the fine particles, and the like. The optical characteristics, flow velocity, spacing, etc. of the detected fine particles are converted into electrical signals and output to the overall control unit (not shown) of the device. The overall control unit controls the frequency of the vibrating element 2 based on this signal, and vibrates the microchip 1 so that the droplets D formed at the orifice 12 contain the fine particles P one by one.

Further, the overall control unit controls the voltage applied to the charged electrode inserted into the charged electrode inlet 20 by synchronizing it with the vibration frequency of the vibrating element 2. As a result, the overall control unit switches between positive and negative charges applied to the sheath liquid and the sample liquid flowing through the sample flow path 11, and applies positive or negative charges to the droplet D formed in the orifice 12. The optical characteristics of the fine particles detected by the optical detection means are converted into an electric signal and output to the overall control unit. The overall control unit controls the voltage applied to the charged electrode based on this signal, and determines the charge to be applied to the droplet according to the optical characteristics of the fine particles contained in each droplet. Specifically, for example, the overall control unit positively charges the droplet containing the fine particles to be sorted with the desired characteristics, and negatively charges the droplet containing the fine particles to be sorted.

At this time, in order to stabilize the charged state of the droplet D, in the fine particle sorting device A, the ground electrodes 6, 6 are provided in the vicinity of the orifice 12 along the moving direction of the droplet discharged into the space outside the chip. Is placed. The ground electrodes 6 and 6 are arranged so as to face each other with the moving droplets interposed therebetween, and are arranged between the counter electrodes 41 and 42 for controlling the moving direction of the fine particles and the orifice 12.

The moving direction of the droplet D charged and discharged from the orifice 12 is controlled by the electric force acting between the counter electrodes 41 and 42. At this time, in order to accurately control the moving direction, it is necessary that a stable electric charge is applied to the droplet. Since a very high voltage is applied to the counter electrodes 41 and 42, when the high potential of the counter electrodes 41 and 42 affects the charge applied to the droplet D from the microtubule 16 at the orifice 12, the liquid The charged state of drop D may become unstable. Therefore, in the fine particle sorting device A, by arranging the grounded electrodes 6 and 6 between the orifice 12 and the counter electrodes 41 and 42, the influence of such a high potential of the counter electrodes 41 and 42 can be exerted. Exclude.

The movement direction of the droplet D discharged from the orifice 12 is controlled, for example, as follows. That is, in the previous example in which the droplet containing the fine particles to be sorted having the desired characteristics is positively charged and the droplet not containing the fine particles to be sorted is negatively charged, the counter electrode 41 is positive and the counter electrode is positive. By negatively charging 42, only the fine particles to be sorted can be sorted into the container 53. Specifically, the droplets containing the fine particles to be sorted, to which a positive charge is applied, move in the direction of arrow f due to the electrical repulsive force with the counter electrode 41 and the electrical attractive force with the counter electrode 42.₃It is controlled in the direction and guided to the container 53. On the other hand, for droplets that are negatively charged and do not contain fine particles to be sorted, the direction of movement is indicated by the arrow f.₂It is controlled in the direction and guided to the container 52.

Alternatively, for example, the droplets containing the particles to be sorted having the desired characteristics are not charged, and the droplets not containing the fine particles to be sorted are positively or negatively charged to form the counter electrodes 41 and 42. When positively or negatively charged, only the fine particles to be sorted can be sorted into the container 51. In addition, the charge applied to the droplet D and the control of the movement direction of the droplet by the counter electrodes 41 and 42 can be performed in various combinations as in the conventional flow cytometry. It should be noted that two or more containers for collecting the droplet D are provided, and the number is not limited to three. Further, these containers may be configured as a discharge channel for discharging the collected droplets without storing them, and the collected fine particles that are not to be sorted may be discarded.

Here, a case has been described in which positive or negative charges are switched and applied to the droplet D based on the characteristics of the fine particles contained in the droplet to perform sorting. The droplets can be separated by charging the droplet D with either positive or negative charges and switching the voltage applied to the counter electrodes 41 and 42 based on the characteristics of the fine particles. .. Further, even when the optical detection means is replaced with an electrical or magnetic detection means, the desired characteristics can be obtained by controlling the moving direction of the droplet in the same manner based on the electrical or magnetic characteristics of the fine particles. The provided fine particles can be collected in any of the containers 51 to 53 and sorted.

As described above, in flow cytometry using a conventional flow cell, the flow cell component that constitutes the flow path system for laminar flow formation and the orifice component for forming droplets are expensive, and each of them is expensive. Since it is necessary to fine-tune (align) the position so that the laminar flow is not disturbed and the configuration is not disposable, there is a risk of cross-contamination between samples. On the other hand, in the microparticle sorter A, laminar flow formation and characteristic detection of microparticles are performed on the microchip 1 that can be used disposablely by integrating the flow cell component and the orifice component, so that a sample between measurements is performed. Cross-contamination does not occur. Further, the conventional alignment becomes unnecessary, and the user can perform the sorting more easily.

In addition, in the fine particle sorting device A, by controlling the moving direction of the fine particles in the space outside the chip, the moving direction of the fine particles can be controlled like the conventional flow cytometry applying μ-TAS. Higher preparative rates can be achieved without having to do so in the flowing liquid. Further, in the fine particle preparative apparatus A, the liquid feeding pressure of the sample liquid and the sheath liquid can be sufficiently increased in the sample flow path 11, and high-frequency droplets can be ejected from the orifice 12 at high speed, which is high. You can get the speed.

A: Fine particle sorter, A₁: Body, A₂: Cover, A₃: Sorting cover, D: Droplets, P: Microparticles, S: Sample liquid layer flow, T: Sheath liquid layer flow, 1,101: Microchip, 1a, 1b: Substrate layer, 11: Sample flow path, 12: Electrode, 121: Microtubule, 13: Conversion flow path, 14: Sheath inlet, 15: Sample inlet, 16: Microtubule, 161: Opening, 17: Narrowing flow path, 18: Suction flow path, 181: Communication port, 19: Suction outlet, 2: Vibrating element, 20: Charged electrode inlet, 3: Optical detection means, 33: Light irradiation part, 4,41,42: Counter electrode, 43: Counter electrode terminal, 51,52,53: Container, 6 : Ground electrode, 63: Ground electrode terminal, 7: Holder, 71: Suction port, 72: Sheath port, 73: Sample port, 74: Connector