JP2006043500A - Particle sorting apparatus and method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a particle sorting and collecting apparatus that is inexpensive and has a simple configuration that does not require an optical detection unit and a high voltage, and that can quickly collect and collect particles at a high concentration without requiring complicated control.
SOLUTION: Means for producing droplets from liquids containing magnetic particles having different magnetization forces; Means for deflecting the traveling direction of magnetic particles according to the magnitude of the magnetization force of the magnetic particles; and droplets corresponding to the deflection amount And a means for collecting particles.
[Selection] Figure 1

Description

  The present invention relates to a particle sorting and collecting device represented by a cell sorter that sorts and collects particles according to the characteristics of biological particles such as cells, cell organs, and microorganisms in a sample solution, and a collecting method thereof.

  As an example of a conventional cell sorter, an apparatus as shown in FIG. 4 has been put into practical use.

  The sample liquid and the sheath liquid, which are cell suspensions, are stored in the sample container 1 and the sheath container 12, respectively, pressurized by a compressor or a nitrogen gas cylinder and a regulator, etc., guided to the nozzle 3, from which it is ejected as a trickle 4 into the atmosphere. Due to the vibration of the vibrator 5 attached to the nozzle 3, the trickle 4 later drops as a droplet 6. The trickle 4 is irradiated with laser light from the laser light source 13, and the scattered light intensity and fluorescence intensity emitted from the cells in the trickle are measured by the photodetector 14.15. From this result, the properties of the cells are analyzed in real time, and according to the result, the charging means (not shown) applies a positive, negative, or zero charge voltage to the fluid, whereby the droplet 6 is positive or negative. Charged negative or zero. High voltage electrostatic deflection plates 16a and 16b are arranged opposite to the drop trajectory of the droplet, and the falling cells are deflected in a direction corresponding to the electric charge and fall into different containers 17, 18, and 19. Collected. Thus, cells can be sorted and collected according to their properties.

  Japanese Patent Publication No. 06-040061 discloses a cell sorter that moves a capture tube that receives particles such as flowing cells and controls the presence or absence of capture.

Y. Baba et al. , "Micro Total Analysis Systems 2002", Kluwer Academic Publishers, pp. In 326-328, magnetophoresis on a chip utilizing the fact that magnetic particles in a continuous flow deviate from the laminar flow direction depending on the particle volume, flow rate, magnetic field gradient, and viscosity by a vertical magnetic field. ) For the separation of magnetic particles.
Japanese Patent Publication No. 06-040061 Y. Baba et al. , "Micro Total Analysis Systems 2002", Kluwer Academic Publishers, pp. 326-328

  As described above, particle separation and collection devices of various methods have been proposed, but those using charged voltage as droplets require an optical detection unit and a high voltage, which increases the size and cost of the device, and Complex control is also required. In addition, the detector that moves the capture tube still requires a detection unit and requires complicated control, and the concentration of the separately collected particles is reduced.

  In the case of separating magnetic particles by magnetophoresis, the concentration of the separately collected particles becomes thin, and it takes time for the separate collection.

  The object of the present invention is to solve the above-mentioned problems, and is inexpensive with a simple configuration that does not require an optical detection unit or high voltage, and quickly separates particles at a high concentration without requiring complicated control. An object of the present invention is to provide a particle separation and collection device that can collect particles.

  In order to achieve the above object, the particle sorting and collecting apparatus according to the present invention comprises means for producing droplets from liquids containing magnetic particles having different magnetizing forces, and the direction of travel of the magnetic particles according to the magnitude of the magnetizing force of the magnetic particles. And means for collecting droplets corresponding to the amount of deflection.

  As described above, the particle sorting and collecting apparatus according to the present invention is capable of sorting and collecting particles quickly at a high concentration without requiring complicated control, while having an inexpensive configuration. In addition, a plurality of kinds of fractionated substances such as nucleic acids and proteins can be transferred to the next process without being concentrated or adsorbed or bound to the magnetic particles, and there is also an effect that movement and detection using the magnetic particles can be facilitated. .

  The particle sorting apparatus of the present invention converts a sample solution containing magnetic particles into droplets, moves the droplets at a determined speed or acceleration, and changes the direction of movement of the droplets within at least the time during which the droplets are moving. By applying a deflecting force to the liquid droplet, the landing point of the liquid droplet is changed, whereby magnetic particles having substantially the same characteristics are separated.

  The method of converting the solution into droplets is to convert the trickle discharged from the nozzles into droplets by applying vibration to the nozzles (discharge ports), or like an inkjet head used in an inkjet printer. It is possible to add ejection energy to the. Examples of the discharge energy include thermal energy. In the case of an inkjet head using thermal energy, bubbles can be ejected from the nozzle by generating thermal bubbles by applying thermal energy with a heater.

  The number of magnetic particles contained in the droplet is preferably one. The reason why it is preferable that the number of magnetic particles included in the droplet is one is that when two or more magnetic particles are included in the liquid, the magnetic particles are displaced by the interaction between the magnetic field and the magnetic particles. This is because the amount is not constant.

  For this purpose, it is preferable that the sample solution is sufficiently diluted and the opening diameter of the nozzle is sufficiently small.

  The amount of deflection of the droplet increases as the time it takes for the droplet to pass through the magnetic field. For example, for example, a magnet that discharges the droplet in the horizontal direction and generates a magnetic field in the moving direction of the droplet is disposed. Is more preferable.

  Most of the deflection amount variation is determined by the deviation of the droplet ejection speed, the droplet weight, and the weight of the enclosing magnetic particles, so the droplet size and ejection speed can be controlled. Furthermore, it can be said that a method of discharging droplets using an inkjet head used for inkjet is preferable from the viewpoint that the droplets can be discharged in the horizontal direction.

(First embodiment)
A particle sorting and collecting apparatus according to a first embodiment of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a diagram showing a schematic configuration of a particle sorting and collecting apparatus according to the first embodiment. The same or similar members as those in the conventional example of FIG.

  The particle sorting and collecting device includes a sample container 1 for storing a sample solution Sa containing a plurality of magnetic particles in which a target substance is hybridized on a magnetic particle, a tube 2 for supplying the sample solution Sa to the nozzle 3, and a vibration to the nozzle 3. It is formed by a nitrogen cylinder (not shown) to which a compressor or a regulator for supplying a vibrator 5 and a sample solution to be added to the nozzle 3 and supplying a pressure for injecting the sample solution as a trickle 4 from the injection port of the nozzle 3 to the sample container. ing.

  In FIG. 1, the nozzle 3 is arranged so that the trickle 4 ejected from the nozzle 3 goes directly below the nozzle, but although not particularly shown, it may be arranged to be ejected horizontally or in another direction. good.

  The trickle 4 ejected from the nozzle 3 is vibrated by the vibrator 5 to the nozzle 3 and becomes a droplet 6 in the middle of dropping due to this vibration.

A separate collection container 8 divided into a plurality of rooms is arranged under the fall path of the trickle 4, and a magnet 7 is placed below the position where the trickle 4 becomes the droplet 6 in the fall path of the trickle 6. Yes. The separate collection container 8 is divided into four chambers A, B, C, and D from directly below the nozzle 3 toward the magnet 7.
Although FIG. 1 shows an example in which the diluted sample liquid Sa is supplied to the nozzle, the configuration is such that the sheath liquid is supplied to the nozzle and the sample liquid Sa is diluted in the nozzle as in FIG. Needless to say, you can.

  Below, the method of fractionating for every dimension of magnetic particles using sample liquid Sa containing magnetic particles of three kinds of dimensions is explained in detail.

  A magnet 7 is disposed around the drop path of the droplet 6, and a sorting collection container 8 is disposed below the nozzle 3. The separation collection container 8 is divided into four chambers A, B, C, and D from directly below the nozzle 3 toward the magnet 7.

  Magnetic particles are generally spherical particles containing a magnetic substance of iron oxide, and many particles having a diameter of 0.1 to 10 μm are commercially available. In this example, three types of magnetic particles b, c, and d having diameters of 4 μm, 4.5 μm, and 5 μm were used. A DNA probe having a different arrangement is bound to each magnetic particle b, c, d. These magnetic particles are subjected to a hybridization reaction with DNA extracted from a specimen such as blood, and are sufficiently diluted with a solution having a salt concentration that does not affect the hybridization reaction. This solution is stored in the sample container 1 as the sample solution Sa.

  Since the sample liquid Sa is sufficiently diluted, the droplet 6 can include not only those containing magnetic particles but also those containing no magnetic particles. The liquid droplets that do not contain magnetic particles fall directly under the influence of the magnetic field by the magnet 7 and enter the room A of the sorting container 8. On the other hand, in the case of a droplet containing the magnetic particles b, c, d, the particle size of the magnetic particles when magnetized is increased according to the size of the magnetic particles, that is, the magnetic particles having a large particle size have a high iron oxide content. Because it has a larger magnetizing force than small magnetic particles, it is attracted to the magnet. As a result, the magnetic particles b, c, and d are deflected so as to approach the magnet in the order, and enter the rooms B, C, and D of the separate collection container 8, respectively.

  At this time, if the particle diameter, iron oxide content, droplet liquid amount, and DNA amount vary, the drop position is slightly shifted in the horizontal direction on the paper surface, but the rooms B, C, and D of the separate collection container 8 are sufficiently wide. Because it has an area, it is surely collected separately. In this way, it is possible to separately collect three types of B, C, and D according to the size of the magnetic particles, that is, according to the DNA sequence.

  The DNA that has undergone hybridization reaction with these separately collected magnetic particles is bound to the magnetic particles, so if it is necessary to concentrate, it can be easily concentrated using another magnet or electromagnet, and a magnetic field is used. You can also move. If necessary, it can be easily removed from the magnetic particles by a solution having a certain salt concentration, heat, etc., and the remaining DNA sequence and DNA amount can be examined or used for other purposes.

  The magnetic particles are not necessarily composed of a single kind of material, and at least one magnetic particle is larger than the magnetic particles and is covered with particles that are hardly affected by the magnetic field, or the magnetic particles are magnetic fields. The particles may be configured to cover particles that are hardly affected by the above. Further, the shape of each particle is not necessarily spherical, and may be a non-uniform shape without steric symmetry.

  In this embodiment, the pressure of the sample container is adjusted and applied so that the flow rate is 2 m / second at the nozzle portion, the nozzle injection diameter is 20 μm, the vibration of 50 KHz is applied to the nozzle, and 0.9 Tesla is applied. A magnet was used. When the distance between the collection container and the injection port is 14 cm, the room of the collection container is 1.1 cm on one side, and the center of the room A is placed directly under the nozzle, so that the magnetic particles of each diameter Were collected separately.

In order not to include two or more magnetic particles in the droplet under these conditions, the sample solution should be diluted so that the number of magnetic particles contained in the sample solution is 4 × 10 ⁷ particles / cc or less. good.
(Example 2)
FIG. 2 is a diagram showing a schematic configuration of the second embodiment. The same or similar members as those in the conventional example of FIG.

  The sample liquid Sa including a plurality of magnetic particles is guided to the droplet discharge nozzle 10 through the tube 9, and the droplet 6 including the individual particles is discharged downward by the droplet discharge nozzle 10 and falls. The droplet discharge nozzle 10 has a well-known configuration as an inkjet head for an inkjet printer. In this embodiment, a thermal energy is applied by a heater to generate bubbles, and a droplet discharge nozzle is used. (See, for example, JP-A-2004-202800).

  An electromagnet 11 is disposed around the drop path of the droplet 6, and a sorting container 8 is disposed below the droplet discharge nozzle 10. The separation collection container 8 is divided into four rooms A, B, C, and D from directly below the droplet discharge nozzle 10 toward the electromagnet 11.

In this example, the opening diameter of the droplet discharge nozzle was set to 20 μm, and the droplet size was controlled to be 8 pl and the discharge speed was 12 m / s. As a result, the same result as in Example 1 was obtained.
(Third embodiment)
In the third embodiment, magnetic particles and a sample using the magnetic particles will be described. As the magnetic particles used in this example, magnetic microspheres having the product names EM1-100 / 20, 30, and 40 manufactured by Merck as shown in FIGS. 3B, 3C, and 3D were used.

  This magnetic particle has a structure in which ferrite is present on the inside and the outside is covered with polystyrene. The size of the magnetic particles is 0.9 μm to 1.8 μm. In FIGS. 3B, 3C, and 3D, the ferrite content is changed to 15 to 25%, 26 to 35%, and 36 to 50%, respectively.

  In addition, a capture body for capturing a target substance is immobilized on each magnetic particle. Here, the magnetic particles shown in FIGS. 3B, 3C, and 3D modified with biotin are modified with streptavidin. Anti-CEA antibody, anti-AFP antibody, and anti-PSA antibody are bound.

  A blood sample was reacted with these magnetic particles. If various antigens of CEA, AFP, and PSA, which are known as cancer markers, are present in the blood sample, they bind to the anti-CEA antibody, anti-AFP antibody, and anti-PSA antibody on the surface of the magnetic particles, respectively, by an antigen-antibody reaction. This reaction solution was sufficiently diluted so that a plurality of magnetic particles were not contained in one droplet when forming the droplet. This solution is guided to the droplet discharge nozzle 10 as the sample liquid Sa.

  Next, the operation of the apparatus of this embodiment will be described. Since the sample liquid Sa is sufficiently diluted, the droplet 6 can include not only those containing magnetic particles but also those containing no magnetic particles. The liquid droplets that do not contain the magnetic particles fall and fall directly under the influence of the magnetic field by the electromagnet 11 and enter the room A of the sorting collection container 8. On the other hand, in the case of a droplet containing any one of the magnetic particles b, c, d, depending on the content of ferrite, that is, the magnetic particles having a high content of ferrite have a ferrite content when magnetized. Since the magnetic particles have a larger magnetizing force than the small magnetic particles and tend to gather in the magnet, the magnetic particles b, c, and d are deflected so as to approach the magnet, and enter the rooms B, C, and D of the separate collection container 8, respectively. .

  At this time, if the particle diameter, the ferrite content, the liquid amount of the droplets, and the antibody amount vary, the dropping position is slightly shifted in the horizontal direction on the paper surface, but the rooms B, C, and D of the separation collection container 8 have a sufficiently large area. Therefore, it is surely collected separately. In this way, it is possible to separately collect B, C, and D according to the ferrite content, that is, according to the type of antigen.

  As a result of separation under the same conditions as in Example 1, substantially the same results as in Example 1 could be obtained.

  The magnetic fine particles classified according to the size of the magnetic particles or the content of the magnetic material in Examples 1 to 3 are diluted in the sample solution, but only the droplets containing the magnetic particles are separated and collected substantially. It is collected in a concentrated state.

  Since these separately collected antigens are bound to magnetic particles, if further concentration is required, they can be easily concentrated by fixing the magnetic particles using another magnet or electromagnet and removing the solution. Moreover, since the movement using a magnetic field can also be performed, the amount of antigens can be examined, and further detailed examination can be performed.

  In the above examples, the substance to be captured by the magnetic particles is DNA extracted from a specimen such as blood and the marker antigen in the blood. However, modification of the magnetic particles can be modified according to the captured substance in RNA and other proteins. Applicable to the surface.

  In the above embodiments, the magnetizing force of the magnetic particles is changed by the same amount of the magnetic material, but the magnetizing force may be changed depending on the shape of the magnetic particles or the magnetic material, or different magnetic materials may be used. The magnetizing force may be changed.

It is a block diagram of the particle | grain fraction collection apparatus of embodiment. It is a block diagram of the particle | grain fraction collection apparatus of another embodiment. It is a block diagram of a magnetic particle. It is a block diagram of a conventional apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample container 2 Tube 3 Nozzle 4 Fine grain 5 Vibrator 6 Droplet 7 Magnet 8 Sorting collection container 9 Tube 10 Droplet discharge nozzle 10
DESCRIPTION OF SYMBOLS 11 Electromagnet 12 Sheath container 13 Laser light source 14, 15 Photo detector 16 Deflection plate 17, 18, 19 Container

Claims (10)

Means for producing a droplet from a liquid containing magnetic particles having different magnetization forces; means for deflecting the traveling direction of the droplet according to the magnitude of the magnetization force of the magnetic particles contained in the droplet; and a liquid according to the deflection amount And a means for collecting droplets. 2. The particle sorting and collecting apparatus according to claim 1, wherein different sorting substances are adsorbed or bonded to the magnetic particles having different magnetizing forces. 3. The particle sorting and collecting apparatus according to claim 1, wherein the magnetic particles having different magnetizing forces are magnetic particles having different sizes. 3. The particle sorting and collecting device according to claim 1, wherein the number of magnetic particles contained in the droplet is one. The particle sorting and collecting apparatus according to claim 1 or 2, wherein the means for producing droplets has a discharge port and discharge energy generating means. A step of making droplets from liquids containing magnetic particles having different magnetization forces, a step of deflecting the traveling direction of the magnetic particles according to the magnitude of the magnetization force of the magnetic particles, and a step of collecting droplets according to the deflection amount A particle fraction collection method comprising: A step of adsorbing or binding different fractional substances to magnetic particles having different magnetizing forces, a step of forming droplets from a liquid containing magnetic particles adsorbed or bound by the fractional substances, and a magnetizing force of magnetic particles contained in the droplets A method for separating and collecting particles, comprising: a step of deflecting a traveling direction of a droplet according to a size; and a step of collecting a droplet according to a deflection amount. The method according to claim 6 or 7, wherein the magnetic particles having different magnetizing forces are magnetic particles having different sizes. 8. The particle sorting and collecting method according to claim 6, wherein the number of magnetic particles contained in the droplet is one. 8. The particle sorting and collecting method according to claim 6 or 7, wherein the droplets are formed by being discharged from a discharge port by the action of discharge energy.

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