JP6755178B2 - Particle manipulation device and particle classification method using the device - Google Patents

Description

The present invention relates to a device for manipulating particles contained in a fluid and a method for manipulating particles contained in a fluid using the device.

When a solid substance composed of an inorganic substance, an organic substance, a metal, etc. is applied as a material to various industrial fields, for example, it is supplied as a powder as a primary raw material, and the powder is formed into spherical particles or formed into another shape. It is supplied as a product. These materials (solids) are designed by examining their physical properties in order to improve the performance of the final product. In addition, there are many examples in which spherical particles are applied as final products, such as crushing balls (alumina, zirconia, silica, etc.), liquid crystal spacers (resin, silica, etc.), chromatographic separators, and other adsorbents. Can be mentioned. In general, such particle products having a uniform shape, size, and density greatly affect the characteristics of the product as a final form, and the current situation is that more uniform particles are required.

As a method for producing such particles, a method for developing a technique for producing uniform particles from the beginning (Patent Documents 1 and 2) and a method for producing particles having a non-uniform distribution with a required size, density, and shape are used. There are two methods of taking out (Patent Documents 3 and 4). The former is a relatively new technology and requires renewal of manufacturing equipment, but the latter can be added to the current equipment and is relatively easy to incorporate into the manufacturing process. As the latter technique, for particles of several tens of μm or more, for example, a filter separation method (including sieve classification), a gravity classification method, a centrifugation method, a cyclone separation method, and the like exist.

The separation or classification method mentioned above is extremely difficult to separate particles of several tens of μm or less, especially particles of 10 μm or less, and it is generally a batch process and it is difficult to continuously separate them. .. In the batch process, the amount of processing at one time is determined by the separation yield, so relatively large equipment (stock container for raw materials, supply equipment, collection container for unnecessary products) is required. For example, the sieve classification method is suitable for mass processing, but it is a batch process because it is necessary to gradually change the size of the sieve mesh. The gravity classification method is basically a batch process similar to the sieve classification method, and it takes an enormous amount of time to process as the particle size becomes smaller. Centrifugation and cyclone separation are suitable for high-speed processing, but require large equipment and are not suitable for continuous processes.

However, if the classification can be performed continuously, it is possible to reduce the amount of the original raw material charged and the amount of production to the utmost limit. Also, in the medical field, there is a step called B / F separation in order to separate the components that have not reacted with the fluorescent substance adsorbed on the particles when inspecting using the antigen-antibody reaction, and such a very small amount of sample Particle separation is also used for inspection. However, most of the particle separations used here use magnetic particles to separate and clean the particles with a magnet, so there is a problem that it takes time for this step.

In addition, as a technique for separating finer particles (DNA, vaccine, etc.), a brownia ratchet, which is a finely processed hook-shaped protrusion, is regularly arranged and the brown vibration of the particles is used to obtain the desired size from the mainstream flow trajectory. There is also a technique for separating (separating) the DNA or vaccine of the above (Non-Patent Document 1). However, since this method is a split flow formation by Brownian motion as the separation mode, the separation rate is slow, and the main flow formation is performed in the form of electrophoresis, which takes time for separation, especially with a diameter of 1 μm or more. It takes a long time to separate particles, and there is a problem that they cannot be put into practical use due to the influence of turbulence during that period.

Further, as a method of separating or classifying cells based on their size, a method of measuring the cell size by measuring forward scattered light using a flow cytometer is known. The flow cytometer can roughly separate the target substance from a large amount of particles, but it is difficult to accurately separate a small amount due to the influence of the cell shape and refractive index, and the purpose is vulnerable to impact. Things can be destroyed.

There are techniques and devices for separating particles of a specific size from particles of various sizes, and examples thereof include a sieve classification method, a gravity classification method, a centrifugation method, and an electrophoresis method. Although each is suitable for batch processing, it is not suitable for continuous classification processing.

Patent No. 3746766 Patent No. 4032128 Patent No. 4760330 Patent No. 4462058 WO2004 / 008132

D0387A Vol.96, NO.23 Proceedings of the National Academy of Sciences of the United States of America PAGE.13165-13169 1999

A first object of the present invention is the development of a particle manipulation device and a particle manipulation method that enable manipulation of particles contained in a fluid. There is a demand for a particle manipulation device and a particle manipulation method that enable manipulation of particles having specific properties from among particles having various properties contained in a fluid. A second object of the present invention is to provide a device for manipulating the particles from a fluid containing the particles, which can be miniaturized, and which can classify the particles by manipulating the particles. There is.

In view of the first problem, the present inventors have applied reciprocating flow to a fluid containing particles having various properties, and the size, shape, mass, density, etc. of the particles have been adjusted. Focusing on the movement with the mobility peculiar to the particles, we have invented a device and a method for manipulating the particles contained in the fluid by reciprocating flow.

Therefore, in the first aspect, the present invention relates to:
[1] A particle manipulation device including a flow path of a fluid containing particles and one or a plurality of pressurizing and depressurizing means for generating a reciprocating flow, in which particles in the fluid move in one direction of the reciprocating flow. The particle manipulation device that enables the movement, separation, or classification of particles by moving with the mobility peculiar to the particles.
[2] The particle manipulation device according to item 1, wherein the reciprocating flow is a periodic flow.
[3] The particle manipulation device according to item 2, wherein the waveform indicating the displacement amount of the fluid in the periodic flow has a non-axisymmetric waveform.
[4] The particle manipulation device according to any one of items 1 to 3, wherein the flow path is formed between the upper surface substrate member, the lower surface substrate member, and the side surface member.
[5] The particle manipulation device according to item 4, wherein the pressurizing / depressurizing means is arranged on one or more members selected from the group consisting of the upper surface substrate member, the lower surface substrate member, and the side surface member.
[6] The particle manipulation device according to item 1 to 5, wherein the flow path includes one or more fluid inlets and one or more fluid outlets.
[7] Item 6 in which the fluid introduction port and the fluid discharge port are arranged so that the flow from the fluid introduction port to the fluid discharge port can be formed and maintained in a direction intersecting the direction of the reciprocating flow. The particle manipulation device according to.
[8] A particle manipulation method comprising a step of applying a reciprocating flow to a fluid containing particles; and a step of moving the particles in one direction of the reciprocating flow with a particle-specific mobility.
[9] The particle manipulation method according to item 8, wherein the reciprocating flow is a periodic flow.
[10] The particle manipulation method according to item 9, wherein the waveform indicating the displacement amount of the fluid in the periodic flow has a non-axisymmetric waveform.
[11] The particle manipulation method according to any one of items 8 to 10, wherein the method includes a step of acquiring the transferred particles and enables classification or separation of the particles.
[12] Item 6 in which the fluid introduction port and the fluid discharge port are arranged so that the flow from the fluid introduction port to the fluid discharge port can be formed and maintained parallel to the direction of the reciprocating flow. The described particle manipulation device.

Further, as a result of diligent studies in order to solve the second problem, the present inventors have reached the following invention from the second viewpoint.
That is, the first aspect of the present invention from the second aspect is
A particle manipulation device comprising a flow path of a fluid containing particles and one or more pressurizing / depressurizing means for generating reciprocating flow.
The pressurizing / depressurizing means is a means for generating a reciprocating flow in a direction intersecting the introduction direction of the fluid in the flow path.
The waveform indicating the displacement amount of the fluid in the reciprocating flow has a non-axisymmetric waveform.
The particle manipulation device is provided with one or more uneven portions in the flow path.

The second aspect of the present invention according to the second aspect is the particle manipulation apparatus according to the first aspect, wherein the flow path is formed between the upper substrate member, the lower substrate member, and the side surface member. Is.

Further, in the third aspect of the present invention from the second aspect, the uneven portion is arranged on either one or both of the upper substrate member and the lower substrate member, and is in a direction parallel to the introduction direction of the fluid in the flow path. The particle manipulation device according to the second aspect, which extends to the above.

The fourth aspect of the present invention from the second aspect is the particle manipulation device according to the third aspect, wherein the uneven portion provided in the flow path extends linearly.

The fifth aspect of the present invention from the second aspect is the particle manipulation device according to the fourth aspect, wherein the cross section of the uneven portion extending linearly has a non-axisymmetric shape.

A sixth aspect of the present invention from the second aspect is the first to fifth means in which the pressurizing / depressurizing means is a means for generating a flow reciprocating in a direction perpendicular to the introduction direction of the fluid in the flow path. The particle manipulation device according to any one of the above embodiments.

Further, in the seventh aspect of the present invention from the second aspect, a plurality of particle discharge ports are provided in the flow path of the fluid containing particles, and the particle discharge ports are provided in the direction perpendicular to the introduction direction of the fluid containing particles. , The particle manipulation device according to any one of the first to sixth aspects.

The eighth aspect of the present invention from the second aspect is the particle manipulation device according to the seventh aspect, wherein particles having different diameters are discharged from each particle discharge port.

A ninth aspect of the present invention from the second aspect is a method of classifying particles contained in a fluid using the particle manipulation device according to any one of the first to eighth aspects.

A tenth aspect of the present invention of the second aspect is
A particle manipulation device comprising a flow path of a fluid containing particles and one or more pressurizing / depressurizing means for generating reciprocating flow.
The pressurizing / depressurizing means is a means for generating a flow reciprocating in a direction parallel to the introduction direction of the fluid in the flow path.
The waveform indicating the displacement amount of the fluid in the reciprocating flow has a non-axisymmetric waveform.
The particle manipulation device is provided with one or more uneven portions in the flow path.

The eleventh aspect of the present invention according to the second aspect is the particle manipulation according to the tenth aspect, wherein the flow path is formed between the upper substrate member, the lower substrate member, and the side surface member. It is a device.

Further, in the twelfth aspect of the present invention from the second aspect, the uneven portion is arranged on either one or both of the upper substrate member and the lower substrate member, and intersects with the introduction direction of the fluid in the flow path. The particle manipulation device according to the eleventh aspect, which extends in the direction.

According to the present invention of the first aspect, particles contained in a fluid can be moved with a unique mobility by giving a reciprocating flow in a direction intersecting or parallel to the flow path, and the particles can be classified. It becomes possible to do.
Further, according to the second aspect of the present invention, in a particle manipulation device including a flow path of a fluid containing particles and one or a plurality of pressurizing / depressurizing means for generating a reciprocating flow, the pressurizing / depressurizing means is the flow. It is a means for generating a reciprocating flow in a direction intersecting or parallel to the introduction direction of the fluid in the path, and the waveform indicating the displacement amount of the fluid in the reciprocating flow has a non-axisymmetric waveform and is in the flow path. It is characterized in that one or a plurality of uneven portions are provided. The particle manipulation device of the present invention of the second aspect can also continuously classify the particles contained in the fluid. Further, since the particle manipulation device of the present invention from the second viewpoint has a simple device configuration, it can be easily miniaturized.

FIG. 1 is a side view of the particle manipulation device of the present invention from the first aspect. FIG. 2A is a diagram schematically showing a side surface of the flow path structure. FIG. 2B is a perspective view of a lower surface substrate, a side surface member, and an upper surface member of a flow path structure having a plurality of inlets and outlets. FIG. 3A is a diagram schematically showing the flow of liquid generated by the flow path being deformed by the vibrating member. FIG. 3B shows the voltage applied to the vibrating members 9L and 9R. FIG. 3C is a diagram showing expansion and contraction of the piezoelectric element when a voltage is applied. FIG. 4A is a diagram schematically showing a side surface of the flow path structure. FIG. 4B is a top view of the side member constituting the flow path structure. The side surface member is a hollowed out silicone rubber sheet having a thickness of 11 mm, and the hollowed out portion is sandwiched between the upper surface substrate and the lower surface substrate to form a flow path. FIG. 5A is a top view of a side member of the flow path structure of FIG. 4, which comprises a flow path structure in which a plurality of fluid discharge ports are provided for one fluid introduction port and the fluid discharge port is used as a particle acquisition port. Is. FIG. 5B is a photograph of the particle manipulation device of the present invention from the first aspect including the flow path structure. FIG. 6 shows a method for calculating the moving speed of particles. Each frame of the image observed by the high-speed camera is binarized to detect the enclosed particles. FIG. 7 shows the mobility of particles when the waveform of the applied voltage is deformed. FIG. 7A shows the mobility when a voltage of a waveform having a positive deformation (0 °, 30 °, 60 °, 120 °, and 150 °) is applied, and FIG. 7B shows a negative deformation (-0 °). , -30 °, -60 °, -120 °, and -150 °), and the mobility when a voltage of a waveform is applied. FIG. 8 shows the mobility when a waveform having a positive deformation is applied, and when a waveform having a deformation of 180 ° (sawtooth wave) is applied, the direction is opposite to that of other waveforms. Indicates that it has moved to. FIG. 9 shows the change in mobility when the solvent for dispersing the particles is replaced with Ficoll from ethanol. FIG. 10A is a diagram schematically showing a side surface of the flow path structure. FIG. 10B is a top view of the side member constituting the flow path structure. The side surface member is a hollowed out silicone rubber sheet having a thickness of 11 mm, and the hollowed out portion is sandwiched between the upper surface substrate and the lower surface substrate to form a flow path. FIG. 11A is a photograph of a flow path structure for cell separation. FIG. 11B is an enlarged view of the flow path, showing that cancer cells and blood cells have been separated. FIG. 12 is a side view of another aspect of the particle manipulation device of the present invention from the first aspect. FIG. 13 is a side view of still another aspect of the particle manipulation device of the present invention from the first aspect. FIG. 14 is a principle diagram of the particle manipulation device of the present invention from the first viewpoint capable of separating / measuring particles in batch units. FIG. 15 is a configuration diagram showing one aspect of the particle manipulation device of the present invention from the first viewpoint, which enables separation / measurement of particles in batch units. The figure which showed an example of the particle manipulation apparatus of this invention of the 2nd viewpoint (side view). Top view of a lower substrate member among the flow path structures constituting the particle manipulation device shown in FIG. Top view of an upper substrate member among the flow path structures constituting the particle manipulation device shown in FIG. Top view of side member of the flow path structure constituting the particle manipulation device shown in FIG. FIG. 6 is a schematic view showing flow generation due to expansion / contraction of a piezoelectric element in the particle manipulation device shown in FIG. The top view of the side surface member among the flow path structures constituting the particle manipulation apparatus produced in Example 6. The schematic diagram which showed the movement of the particle in Example 6. The figure which showed the result of Example 7. Black circles or black squares are the result of particles of φ200 μm, and white circles and white squares are the result of particles of φ80 μm. The figure which showed the result of Example 8. Black circles or white circles are the result of particles of φ200 μm, white and white squares are the result of particles of φ80 μm, and black and white triangles are the result of particles of φ40 μm. The schematic diagram which showed the movement of the particle in the comparative example 1. FIG. The figure which showed an example of the lower substrate member, the upper substrate member, and the side surface member of the flow path structure which enables continuous classification in the particle manipulation apparatus of this invention of the 2nd viewpoint.

The present invention of the first aspect relates to a particle manipulation device including a flow path of a fluid containing particles and one or more pressurizing and depressurizing means for generating reciprocating flow. The present invention of the second aspect relates to a particle manipulation device including a flow path of a fluid containing particles and a pressurizing / depressurizing means for generating a flow reciprocating in a direction intersecting the introduction direction of the fluid in the flow path. .. In both respects, the particle manipulation device of the present invention is a device that moves particles according to particle-specific properties, such as, but not limited to, size, density, shape, mass, and the like. The particle manipulation device of the present invention is based on the fact that particles in a fluid contained in a flow path move in one direction of the reciprocating flow by generating a reciprocating flow with a mobility peculiar to the particles. It allows the particles to be manipulated, i.e. move, separate, or classify. Since the mobility peculiar to particles changes depending on various conditions, a desired particle group can be selected as conditions such as a fluid, a flow path length, a position of a particle acquisition port, and a waveform indicating a displacement amount of the fluid. Will be able to be obtained.

The particles to be transferred, separated, or classified in the present invention are not particularly limited as long as they are substances insoluble in the fluid, such as beads, crushing balls, liquid crystal spacers, chromatographic separating agents, and adsorbents. In addition to industrial materials, research and medical materials such as cells, DNA, vaccines, viruses, colloids, and micelles are also included. In the present invention, since separation is performed by the reciprocating flow of a fluid, separation is possible regardless of electrostatic charge or dielectric properties, and fragile particles such as cells, droplets, bubbles, separation substrate, catalyst, and pharmaceutical granules can be separated. , Capsules, etc. are more suitable for separation.

Examples of cells to be migrated, separated or classified include arbitrary cells, and for example, cells in blood and cultured cells are subjected to migration, separation or classification. Cells in blood are roughly classified into red blood cells, white blood cells and platelets as blood cells, but other cells such as cancer cells may also be present. The size of red blood cells is about 7 to 8 μm in diameter and about 2 μm in thickness, and occupies most of the blood cells. White blood cells include monocytes, lymphocytes (T cells, B cells, natural killer cells, etc.), neutrophils, eosinophils, and basophils, and their size is about 6 to 30 μm depending on the cell type. is there. Platelets are about 1 to 4 μm. On the other hand, it is known that the size of circulating tumor cells (CTC) in blood, which circulates along the flow of blood and lymph and causes metastasis to distant organs, is 20 μm to 200 μm, which is considerably larger than that of blood cells. .. However, the proportion of CTC present in blood is extremely low, and only a few to several tens of CTCs are present per 10 ml of blood, and its detection and separation are difficult. In the present invention, it is also possible to separate cells according to their size, which is effective for detecting and separating CTC from a group of cells existing in blood. In addition, when the cultured cells are separated, for example, when differentiation is induced from stem cells, a desired differentiated cell group can be separated and obtained according to the size.

As mentioned above, in the first aspect, the present invention provides the following particle manipulation devices:
[1] A particle manipulation device including a flow path of a fluid containing particles and one or a plurality of pressurizing and depressurizing means for generating a reciprocating flow, in which particles in the fluid move in one direction of the reciprocating flow. The particle manipulation device that enables the movement, separation, or classification of particles by moving with the mobility peculiar to the particles.

In the present invention of the first aspect, the flow path is provided with a fluid introduction port and a fluid discharge port, and a fluid can flow from the fluid introduction port to the fluid discharge port. The direction in which the fluid flows is defined as the mainstream direction, and the flow is defined as the mainstream. During the classification, the fluid outlet can be plugged, and more preferably the fluid inlet can be plugged to keep the fluid in the flow path. By keeping the liquid in the flow path and operating the particle manipulation device of the present invention, the particles can be manipulated in batch units. In order to acquire the particles classified by the batch unit processing, a plurality of particle acquisition ports may be provided. On the other hand, the particle manipulation device of the present invention can be operated in a state where the fluid is continuously flowing from the fluid introduction port to the fluid discharge port, whereby the particles can be continuously classified. The fluid discharge port of the flow path may be used as the particle acquisition port, or the particle acquisition port may be formed separately from the fluid discharge port. The size of the particle acquisition port may be the same as or different from the size of the liquid discharge port, but from the viewpoint of not disturbing the mainstream flow, 1/2, more preferably 1 / of the size of the fluid discharge port. It is 5, and more preferably 1/10. When forming a planar flow path, a plurality of fluid introduction ports and a plurality of fluid discharge ports may be provided from the viewpoint of reducing turbulence in the mainstream. When classifying particles continuously, the reciprocating flow is applied in the direction intersecting the mainstream direction. The direction intersecting the mainstream direction means that the reciprocating flow direction and the mainstream direction are not parallel to each other. Therefore, the reciprocating flow direction is applied to the mainstream direction and a direction at an arbitrary angle, more preferably a direction orthogonal to the mainstream direction. A particle manipulation device that enables continuous classification is more preferable in that the device configuration can be reduced as compared with a batch type particle manipulation device.
When classifying particles in batch units, the reciprocating flow may be applied parallel to the mainstream direction. Particles can be classified in the same manner as in chromatography by feeding the liquid from the fluid inlet at a constant flow rate, allowing it to stand still, and then applying a reciprocating flow parallel to the mainstream direction. The above aspect is preferable in that even particles having a size of several tens of μm or less, which are difficult to classify by a conventional method, can be easily classified and measured.

The shape of the flow path may be any shape such as tubular and planar. When manipulating particles in batch units, tubular channels may be used. On the other hand, from the viewpoint of continuously acquiring the classified particles, it is preferable to form the flow path in a planar shape and to provide a plurality of particle acquisition ports for the flow path while forming the mainstream. The flow path formed in such a planar shape may be formed between, for example, an upper surface substrate member, a lower surface substrate member, and further a side surface member. The planarly formed flow path may have a shape that branches the reciprocating flow or a shape that collects particles that have moved with a certain range of unique mobility in order to enhance the separation ability due to the reciprocating flow. .. As the upper surface substrate member and the lower surface substrate member, any rigid flat plate can be used, but from the viewpoint of observing particles, a transparent member, preferably a glass plate, a polycarbonate plate, or the like is used. The upper surface substrate member and the lower surface substrate member may have any shape, but the same shape is preferable, for example, a quadrangle. The side surface member may be a rigid member or a flexible or stretchable member. From the viewpoint of applying a vibrating member to generate a reciprocating flow, a flexible or stretchable member is preferable, and for example, silicone rubber, fluororubber, PDMS, elastomer resin, polymer gel, urethane resin and the like are used. The side surface member is sandwiched between the upper surface substrate member and the lower surface substrate member to form a flow path. Therefore, from the viewpoint of reducing the leakage of fluid from the flow path, the side surface member is preferably formed by hollowing out the central portion of the plate-shaped member into an appropriate shape.

The length of the flow path can be appropriately selected depending on the required separation ability of the particles, the properties of the particles to be separated and / or the properties of the fluid flowing through the flow path and the flow velocity. For example, when particles having a particle size of about 200 μm are continuously classified, those having a length of 5 cm or more are used from the viewpoint of achieving high resolution, and particles having a length of 10 cm or more may be used. Further, when classifying on an industrial scale, it may be 30 cm or more. On the other hand, from the viewpoint of using the rigid upper surface substrate member and the lower surface substrate member, it is preferably 10 cm or less, more preferably 5 cm or less. On the other hand, from the viewpoint of downsizing the classifier and reducing the separation time, one having a size of 1 cm or less may be used, but it is not intended to be limited to these numerical values. In addition, when classifying or measuring in batch units, the length of the separation time is proportional to the separation capacity, so it is preferable to appropriately select the length of the flow path within the range in which liquid feeding and reciprocating flow can be generated. ..

The fluid inlet is not particularly limited as long as the fluid can be introduced, but one or more fluid inlets may be formed. The fluid inlet may be connected to the liquid feed pump via a liquid feed tube. When a planar formed flow path is used, it is preferable to provide a plurality of fluid inlets from the viewpoint of reducing turbulence in the mainstream. When a plurality of fluid introduction ports are provided, particles to be separated may be introduced from all of them, but from the viewpoint of classifying the particles according to the mobility in the direction of intersection with the flow path from the fluid introduction port, the particles are introduced. The fluid introduction port to be introduced is preferably a part, more preferably one place, and it is preferable that only the fluid containing no particles is introduced from the other fluid introduction ports. When the planar flow path is formed, the fluid introduction port may be formed in any one or more of the upper surface substrate member, the lower surface substrate member, and / or the side surface member, for example, the introduction port may be formed on the upper surface substrate member. It can also be provided.

The fluid discharge port is not particularly limited as long as the fluid can be discharged, but one or more discharge ports may be formed. The fluid discharge port may be connected to a drain tank or a suction pump via a drain tube, or a detector capable of detecting the passage of separated particles or a particle size size detector is connected. It may also function as a particle acquisition port, in which case the liquid containing the particles is fractionated through or directly through the particle acquisition tube. When the flow path formed in a planar shape is used, a plurality of fluid inlets may be provided, and the same number or different number of fluid discharge ports may be provided from the viewpoint of reducing the turbulence of the mainstream. When a planar flow path is formed between the upper surface substrate member, the lower surface substrate member, and the side surface member, the fluid discharge port is formed in any one or more of the upper surface substrate member, the lower surface substrate member, and the side surface member. It may be provided on the lower surface substrate member or the side surface member, particularly on the lower surface substrate member.

The particle manipulation device of the present invention according to the first aspect includes a pressurizing / depressurizing means for generating a flow reciprocating in a direction intersecting or parallel to a mainstream direction. As the pressurizing / depressurizing means, for example, a vibrating means such as an actuator or a piezoelectric element may be used, a liquid flow or an air flow generating means such as a pump may be used, or a flow reciprocating separately from the flow path for separating particles. A generator may be installed. When the vibrating means integrated with the flow path is used, the reciprocating flow is formed by deforming the flow path by the vibrating means. When the flow path is composed of the upper surface substrate member, the lower surface substrate member, and / or the side surface member, the vibration means is provided on any one or more of the upper surface substrate member, the lower surface substrate member, and / or the side surface member. When means for the upper surface substrate member, the lower surface substrate member, and / or the side surface member are provided, a reciprocating flow may be formed by deformation of each member such as deflection. In a more preferred embodiment, a stretchable member is used as the side surface member, and a vibrating means is provided on the upper surface substrate member or the lower surface substrate member to vibrate the side surface end of any of the substrate members up and down to reciprocate the liquid flow. Occurs. In a preferred embodiment, a reciprocating liquid flow is generated by vibrating one side end of the top substrate member up and down, or by alternately vibrating two or more side ends up and down by two or more vibrating means. Can be done. When two or more vibrating means are used, the frequencies may be the same or different. When a liquid flow or air flow generating means such as a pump is used, for example, even if a plurality of pump connection holes are arranged on one side surface member and the connection holes are connected to the pump to alternately apply positive pressure and negative pressure. Good. In another aspect, a plurality of pump connection holes may be arranged on the side surface members on both sides, and the connection holes may be connected to the pump to alternately apply positive pressure. The plurality of pump connection holes arranged on one side member may be connected to one pump or may be connected to a plurality of pumps.

The reciprocating flow generated by the pressurizing / depressurizing means is preferably a periodic flow. It is preferable that the waveform showing the displacement amount of the fluid in such a periodic flow has a non-axisymmetric waveform. In one aspect, these reciprocating flows can be generated by using a drive signal having a reciprocating waveform as the drive signal of the vibrating means. Therefore, the waveform indicating the displacement amount of the fluid may directly correspond to the waveform of the drive signal of the vibrating means. The drive signal having a reciprocating waveform may be either a voltage or a current. Such a signal may be a pulse that changes sharply in a short time, or may be a continuous wave that changes continuously. From the viewpoint of ensuring the reproducibility of the resolution, such a waveform is preferably periodic. Since a reciprocating flow is generated according to the waveform of the drive signal, if the drive signal is periodic, the reciprocating flow is also periodic. As such a periodic waveform, any waveform can be mentioned, and it may be a point-symmetrical waveform, a non-point-symmetrical waveform, a line-symmetrical waveform, or a non-line-symmetrical waveform. Point symmetry means that the waveform is symmetric with respect to the point where the signal strength becomes 0. In addition, line symmetry means that the signal is reversed in polarity every half cycle of the shortest repetition period, and in the case of line symmetry, the positive signal value and the negative signal value are symmetric with respect to the time axis. It has become. While some waveforms are point-symmetrical and line-symmetrical, there are also point-symmetrical and line-symmetrical waveforms. Examples of waveforms that are point-symmetrical and line-symmetrical include sine waves, square waves, and triangular waves. An example of a waveform that is point-symmetrical but non-axisymmetric is a sawtooth wave. However, it is not limited to these examples, and includes any synthetic waves included in the definition. From the viewpoint of moving particles, it is preferable that the reciprocating flow has a non-axisymmetric waveform.

In the present invention of the first aspect, the fluid flowing through the flow path includes a liquid or a gas, but a liquid is preferable from the viewpoint of achieving higher separability. When a liquid is used, any liquid can be selected depending on the particles to be separated. When the particles to be separated are industrial materials, the solvent used in the production may be used as it is, or the particles may be replaced with an inexpensive and harmless solvent such as water and used in the particle manipulation apparatus. When the particles to be separated are biological materials such as cells, viruses, and antibodies, it is preferable to use a liquid in which these particles are originally dispersed, and especially when cells are used, from the viewpoint of ensuring the survival of the cells. , Culture medium, blood, plasma, physiological saline (PBS, TBS, etc.) can be used. For these liquids, any excipients such as pH adjusters, stabilizers, thickeners, preservatives, antibiotics and the like can be used. From the viewpoint of enhancing the separability, a liquid having an appropriate viscosity can be selected according to the size, density and shape of the particles. When a gas is used as the fluid, it may be preferable to use a pressure generating means in addition to using a vibrating means as a means for generating a reciprocating flow.

Hereinafter, the present invention of the first aspect will be described in detail based on the embodiments shown in the drawings. The particle manipulation device of the present invention according to the first aspect is realized by, for example, the following flow path structure 2, the reciprocating liquid flow generator 1, and the liquid feed pump 3 (FIG. 1). Further, particle observation devices 7 and 8 may be installed to confirm that the particles are properly separated. The method of forming the periodic reciprocating liquid flows 26 and 27 used in the particle manipulation apparatus of the present invention from the first aspect will also be described in detail below.

In the aspect of the flow path structure 1, the flow path structure 2 is formed by the upper surface substrate member 18, the lower surface substrate member 20, and the side surface member 19 (FIG. 2A). An introduction port 22 and an discharge port 23 are provided in either the upper surface substrate member 18 or the lower surface substrate member 20, and the direction from the introduction port 22 to the discharge port 23 is referred to as a flow path direction or a mainstream direction (FIG. 2). The particle acquisition port 25 can be arranged at any position on the upper surface substrate member 18 or the lower surface substrate member 20 (FIG. 2). The vibrating member bonding point 21 is arranged on the non-flow path surface of the upper surface substrate member 18 (FIG. 2B). A plurality of vibrating member bonding points 21 may be provided, preferably two. The straight line connecting the two vibrating member bonding points 21 may preferably intersect with respect to the mainstream direction, and the two vibrating member bonding points 21 are preferably arranged so as to be perpendicular to each other. More preferably, the two vibrating member bonding points 21 are arranged at equal positions with the center of gravity of the upper surface substrate member 18 interposed therebetween. The side surface member 19 may be any member as long as it can form a flow path, but a stretchable member can be preferably used. The side surface member 19 may be a rubber sheet in which a portion serving as a flow path 24 is hollowed out, or may be a rubber packing provided on the surface of the lower surface substrate member. By placing the side surface member 19 on the lower surface substrate member 20 and further mounting the upper surface substrate member 18, the flow path 24 of the present invention from the first aspect is formed in the region sandwiched between these members.

The vibrating flow forming device reciprocating liquid flow generating device 1 may include a substrate holder 11 having a mechanism for holding the flow path structure 2, and vibrating members 9L and 9R (FIG. 1). The vibrating members 9L and 9R can be connected to the vibrating member driving power supplies 12L and 12R that drive the vibrating members 9L and 9R via the vibrating member driving signal outputs 13L and 13R. The vibrating member drive power supplies 12L and 12R may be further connected to the waveform generator 15 via the waveform outputs 16L and 16R. The number of vibrating members may be one, but a plurality of vibrating members may be arranged. For example, when the vibration member of 2 is used, the vibration waveforms may or may not be synchronized. A waveform that is line-symmetrical with respect to the time axis can also be used (Fig. 3).
Further, in the vibrating members 9L and 9R of the reciprocating liquid flow generator 1, the diaphragm flow path 30 vibrated by the vibrating member is connected to the introduction port and the discharge port of the particle separation flow path 31, and the particle separation flow path 31
If a reciprocating liquid flow can be generated inside, the particles may be arranged separately from the particle separation flow path 31 (FIG. 12). Further, when the vibrating member is arranged separately from the particle separation flow path 31, only one vibrating member 9L may be arranged (FIG. 13). In the reciprocating liquid flow generator 1 shown in FIGS. 12 and 13, the reciprocating liquid flow is connected to the introduction / discharge port 47 installed in the diaphragm flow path 30 and the introduction / discharge port 48 connected to the particle separation flow path 31. It is preferable to install a check valve that limits the flow direction so as not to hinder the formation of the particles, or an electric valve that is synchronized with the vibrating member 9L.

Particle observation device A particle observation device including a zoom lens 8 and a camera 7 capable of observing the central portion of the flow path structure can be installed in the vibration flow forming device (FIG. 1). As a result, the movement of particles can be monitored, and an appropriate frequency, flow velocity, etc. can be set.

Method for Forming Reciprocating Liquid Flows The reciprocating liquid flows 26 and 27 are generated when the vibrating members 9L and 9R push up and down the upper surface substrate member (FIG. 3A).
If the particle separation flow path 31 is connected so as to form a vibration flow, as shown in FIGS. 12 and 13, a diaphragm flow path that pushes up and down the vibrating members 9L, 9R and the upper surface substrate portion. 30 may be separated from the particle separation flow path 31.

Method of driving the vibrating member As a method of forming a reciprocating liquid flow, a piezoelectric element can be used as the vibrating members 9L and 9R, and a piezo driver can be used as the vibrating member driving power supplies 12L and 12R. The output drive member drive signal output 13L of the vibration member drive power supply 12L is connected to the vibration member 9L, and the output drive member drive signal output 8R of the vibration member drive power supply 13R is connected to the vibration member 9R. Further, the waveform outputs 16L and 16R of the waveform signal generator 15 are connected to the input vibration member drive source signal inputs 14L and 14R of the vibration member drive power supplies 12L and 12R (FIG. 1).
In the formation of the reciprocating liquid flow, the waveforms of the waveform outputs 16L and 16R of the waveform signal generator 15 can be alternately driven to expand and contract the piezo element by shifting the phase by 180 degrees or inverting the signal. , It may form a reciprocating liquid flow (Fig. 3).
The waveform output 16L of the waveform signal generator 15 goes to the vibration member drive source signal input 14L of the vibration member drive power supply 12L, and the waveform output 16R of the waveform signal generator 15 goes to the vibration member drive source signal input 14R of the vibration member drive power supply 12R. Connecting. The output waveform of the waveform signal generator 15 can be arbitrarily generated, and may be, for example, a point-symmetrical or non-point-symmetrical waveform and / or a line-symmetrical or non-line-symmetrical waveform. Typical examples are sine wave, triangle wave, square wave, trapezoidal wave, and sawtooth wave. The output waveform of the waveform signal generator becomes the waveform of the reciprocating liquid flow in the flow path. From the viewpoint of obtaining higher resolution, a sawtooth wave having a non-axisymmetric waveform is preferable.

Particle introduction / discharge method The introduction port 22 and / or the discharge port 23 are installed by forming a through hole having a diameter of 1 mm in the upper surface substrate member 18 or the lower surface substrate member 20 in the flow path structure 2 (FIG. 2B). .. An eyelet having an outer diameter of 1 mm is fixed to this through hole with an adhesive, and the eyelet and the liquid feed pump 3 are connected by a liquid feed tube 5 to introduce and discharge particles 28 (FIG. 1). When collecting the particles 28 that have moved in the direction intersecting the mainstream direction, it is desirable to add a through hole, which is a particle acquisition port 25, to the lower surface substrate member 20 or the upper surface substrate member 18 in addition to the discharge port ( 2B, 5).

Particle Observation Method Observation and measurement of the movement state of particles due to the oscillating flow may be carried out by the camera 5 and the zoom lens 6 of FIG. It is desirable that the camera 5 be used by switching between a high-speed camera (VFC-1000 manufactured by Tomoei) when observing the vibration of particles and a CCD camera (XCD-V50) manufactured by Sony when observing at low speed. In order to observe the particles, the upper surface substrate member 18 is preferably made of a transparent member, for example, glass or polycarbonate.

Calculation method of moving speed of particles Binarization processing is performed on each image of one frame of the observation video by the high-speed camera of the camera 5, and the enclosed particles are detected. Software for measuring the position of the detected particle with respect to the center of gravity (FIG. 6) was produced by LabVIEW (trade name). By converting this information into data as a change over time, the movement trajectory of the particles is measured.

As mentioned above, in the second aspect, the present invention provides the following particle manipulation devices:
A particle manipulation device comprising a flow path of a fluid containing particles and one or more pressurizing / depressurizing means for generating reciprocating flow.
The pressurizing / depressurizing means is a means for generating a reciprocating flow in a direction intersecting or parallel to the introduction direction of the fluid in the flow path.
The waveform indicating the displacement amount of the fluid in the reciprocating flow has a non-axisymmetric waveform.
The particle manipulation device, which is provided with one or more uneven portions in the flow path.

As one aspect of the classification operation of the particles contained in the fluid using the particle manipulation device of the present invention of the second aspect, the discharge port side of the flow path of the fluid containing the particles is closed, and more preferably the introduction port side is also closed. After closing and retaining the fluid containing the particles in the flow path, there is an operation of moving / classifying the particles in batch units by generating a flow that reciprocates in the direction or parallel to the flow path. When the flow path of the fluid containing particles is provided with multiple discharge ports, particles having different diameters (classified) can be collected from each discharge port by opening the discharge port after the above-mentioned batch unit operation. Can be done. As another embodiment of the classification operation of the particles contained in the fluid using the particle manipulation device of the present invention, the fluid containing the particles is continuously introduced from the introduction port side of the flow path and intersects the flow path. By continuously generating a flow that reciprocates in a direction or in parallel, particles contained in the fluid can be continuously classified. By enabling continuous classification, the device configuration of the particle manipulation device can be reduced as compared with the case of operating in batch units.

The particle outlet may be the same outlet as the fluid outlet, or may be a different outlet. When the particle discharge port is provided as a discharge port different from the fluid discharge port, the diameter is 1/2 or less of the diameter of the fluid discharge port from the viewpoint of not disturbing the flow of the flow path of the particles contained in the fluid. It is preferable that the diameter is 1/5 or less, and more preferably 1/10 or less. When the flow path of the fluid containing particles is planar, it is preferable to provide a plurality of fluid introduction ports and a plurality of fluid discharge ports from the viewpoint of reducing the turbulence of the flow of the flow path.

The shape of the flow path of the fluid containing the particles provided in the particle manipulation device of the present invention according to the second aspect is not limited, and may be tubular or planar. However, when the particle manipulation device of the present invention from the second aspect is a device for continuously classifying particles contained in a fluid, it is preferable to provide a planar flow path and a plurality of particle discharge ports. As an example of the planar flow path, there is a flow path formed between the upper substrate member, the lower substrate member, and the side surface member. The planar flow path has a shape that branches the reciprocating flow and a shape that collects particles that have moved (classified) with a certain range of unique mobility in order to enhance the separation ability due to the reciprocating flow. May be. As the upper substrate member and the lower substrate member, any rigid flat plate can be used, but from the viewpoint of observing particles, it is preferable to use a transparent material such as a glass plate or a polycarbonate plate. The upper substrate member and the lower substrate member may have any shape, but it is preferable that they have the same shape (for example, the same quadrangle as each other). The side surface member may be a rigid member or a flexible or stretchable member, but when the flexible or stretchable member is used as the side surface member, the upper substrate member and the lower substrate member are used. It is preferable in that a reciprocating flow can be generated by vibrating in the vertical direction. As an example of the flexible or stretchable member, silicone rubber, fluororubber, PDMS, elastomer resin, polymer gel, urethane resin can be exemplified. The side surface member forms a flow path by being sandwiched between the upper substrate member and the lower substrate member. Therefore, from the viewpoint of reducing the leakage of fluid from the flow path, the side surface member is preferably manufactured by hollowing out the central portion of the plate-shaped member into an appropriate shape.

The length of the flow path of the fluid containing the particles provided in the particle manipulation device of the present invention of the second aspect determines the required separation ability of the particles, the nature of the particles to be separated, and / or the flow path. It may be appropriately selected according to the properties of the fluid and the flow velocity. For example, when classifying particles having a particle size of about 200 μm with high resolution, the length may be 5 cm or more, and may be 10 cm or more. When classifying on an industrial scale, the length may be 30 cm or more. From the viewpoint of maintaining the rigidity of the upper substrate member and the lower substrate member, the length is preferably 10 cm or less, and more preferably 5 cm or less. If the purpose is to reduce the size of the particle manipulation device or shorten the separation time, the length may be 1 cm or less.

The introduction port of the flow path of the fluid containing particles provided in the particle manipulation device of the second aspect of the present invention is not particularly limited as long as the fluid can be introduced, and the number of introduction ports may be one. There may be more than one. Further, the inlet of the flow path may be connected to the liquid feed pump via the liquid feed tube. When the flow path of the fluid containing particles is formed in a planar shape, it is preferable to provide a plurality of introduction ports from the viewpoint of reducing the turbulence of the flow of the flow path. When a plurality of introduction ports are provided, a fluid containing particles to be separated may be introduced from all the introduction ports, but the particles are moved / reciprocated by a flow reciprocating in a direction intersecting the introduction direction of the fluid in the flow path. From the viewpoint of classification, it is preferable that the introduction port for introducing the fluid containing particles is a part of a plurality of introduction ports, and it is preferable that the introduction port is further limited to one place. It is only necessary to introduce the solvent into the introduction port other than the introduction port into which the fluid containing particles is introduced. When the flow path of the fluid containing particles is formed in a planar shape, the introduction port may be formed in any one or more of the upper substrate member, the lower substrate member, and the side surface member. For example, the introduction port may be formed in the upper substrate member. An embodiment of the provision can be exemplified.

The discharge port of the fluid containing the particles provided in the particle manipulation device of the second aspect of the present invention is not particularly limited as long as the fluid can be discharged, and the number of discharge ports may be one. There may be more than one. The fluid outlet may be connected to the drain tank or suction pump via a drain tube, or when used in combination with a particle outlet, via a particle acquisition tube or directly. The fluid containing the particles may be recovered from the water. When the flow path of the fluid containing particles is formed in a planar shape, it is preferable to provide a plurality of discharge ports as well as the introduction port from the viewpoint of reducing the turbulence of the flow of the flow path. The number of outlets installed may be the same as the number of inlets, or may be different. When the flow path of the fluid containing particles is formed in a planar shape, the discharge port may be formed on any one or more of the upper substrate member, the lower substrate member, and the side surface member. For example, the discharge port may be formed on the lower substrate member. An embodiment of the provision can be exemplified (Fig. 26).

Acceleration / depressurization for moving the particles by generating a flow reciprocating in a direction intersecting with or parallel to the introduction direction of the fluid in the flow path of the fluid containing the particles provided in the particle manipulation device of the second aspect of the present invention. As an example of the means, there is a means for moving the particles by generating a reciprocating flow by using a vibrating means such as an actuator or a piezoelectric element or a liquid flow generating means such as a pump. When the flow path of the fluid containing particles is composed of an upper substrate member, a lower substrate member and a side surface member, and a reciprocating flow is generated by using a vibrating means, the vibrating means is applied to any one or more of the above-mentioned members. It may be provided and a reciprocating flow may be generated by deforming each member by the vibrating means. In particular, when the side surface member is a flexible or stretchable member, a vibrating means is provided on the upper substrate member or the lower substrate member, and the side surface end of the member provided with the vibrating means is vibrated up and down to reciprocate the flow. Should be generated. In a preferred embodiment, reciprocating flow can be generated by vibrating one side edge of the upper substrate member up and down, or by alternately vibrating two or more side edges up and down by two or more vibrating means. it can. When a plurality of vibrating means are provided, the frequencies may be the same or different. When generating a reciprocating flow using a liquid flow or an air flow generating means such as a pump, for example, a plurality of pump connection holes are arranged on one side member, and a positive pressure is applied with the connection holes connected to the pump. Flow may be generated by alternately applying negative pressure. Further, a plurality of pump connection holes may be arranged on the side surface members on both sides, and the flow may be generated by alternately applying positive pressure while the connection holes are connected to the pump. A plurality of pump connection holes arranged on one side member may be connected to one pump or may be connected to a plurality of pumps. The direction of the reciprocating flow generated by the pressurizing / depressurizing means may be a direction intersecting or parallel to the introduction direction of the fluid in the flow path of the fluid containing particles, but a direction intersecting the introduction direction of the fluid. In the case of, it is preferable to make it vertical.

The reciprocating flow formed by the method described above is preferably a periodic flow. When the flow is generated by the vibrating means, it can be generated by using a drive signal having a reciprocating waveform as the drive signal of the vibrating means. Therefore, it can be said that the waveform indicating the displacement amount of the fluid directly corresponds to the waveform of the drive signal of the vibrating means. The drive signal having a reciprocating waveform may be either a voltage or a current. The drive signal may be a pulse that changes sharply in a short time, or may be a continuous wave that changes continuously. From the viewpoint of ensuring the reproducibility of the resolution, such a waveform is preferably periodic. Since a reciprocating flow is generated according to the waveform of the drive signal, if the drive signal is periodic, the reciprocating flow is also periodic. In the particle manipulation device of the present invention from the second viewpoint, the reciprocating flow waveform is a waveform having a non-axisymmetric waveform such as a sawtooth type wave, and by using this waveform, before and after switching the traveling direction of the flow. Since the speeds are different, the particles contained in the fluid can be moved efficiently. Here, line symmetry means that the signal is reversed in polarity every half cycle of the shortest repetition period, and in the case of line symmetry, the positive signal value and the negative signal value are symmetric with respect to the time axis. There is. The reciprocating flow waveform used in the particle manipulation device of the present invention may have a non-axisymmetric waveform, and may be a composite wave with other waves (sine wave, triangular wave, square wave, etc.). Good.

Examples of the fluid to be introduced in the particle manipulation apparatus of the second aspect of the present invention include liquids and gases, but liquids are preferable in that higher resolution can be achieved. When a liquid is used, it can be appropriately selected depending on the particles to be separated. When the particles to be separated are industrial materials, the solvent used at the time of production may be used as it is as the introduction liquid, or a liquid replaced with an inexpensive and harmless solvent such as water may be used. When the particles to be separated are biological materials such as cells, viruses, and antibodies, it is preferable to use a solvent in which the biological materials are dispersed, and particularly when cells are used, a culture medium is used from the viewpoint of ensuring cell survival. , Blood, plasma, physiological saline (PBS (Phosphate Buffered Saline), TBS (Tris Buffered Saline), etc.) may be used as a solvent. These liquids may further contain any excipients such as pH adjusters, stabilizers, thickeners, preservatives, antibiotics and the like. From the viewpoint of enhancing the separability, a liquid having an appropriate viscosity may be selected according to the size, density and shape of the particles.

The particle manipulation device of the present invention according to the second aspect is characterized in that one or a plurality of uneven portions are provided in the flow path of the fluid containing the particles. In the present invention, the uneven portion may be composed of only the convex portion, may be composed of only the concave portion, or may be composed of a combination of the concave portion and the convex portion. The shape of the uneven portion is not particularly limited, and examples thereof include a conical shape, a cylindrical shape, a pyramid shape, and a prismatic shape. Further, the uneven portion may extend linearly or curvedly in one direction. When the uneven portion extends, the cross section may have any shape, for example, a point-symmetrical shape, a non-point-symmetrical shape, a line-symmetrical shape, or a non-line-symmetrical shape. Among them, as the uneven portion provided in the flow path of the fluid containing particles, the uneven portion extending linearly and having a non-axisymmetric cross section is preferable, and the uneven portion extending linearly with non-axisymmetry such as a sawtooth shape. It is more preferable to adopt the concavo-convex portion, in that the particles contained in the fluid can efficiently move in one direction. The position where the unevenness is provided is not particularly limited, and may be provided in a direction parallel to the introduction direction of the fluid, or may be provided in a crossing direction, for example, a vertical direction, but the pressurizing / depressurizing means is provided in the flow path. When it is a means for generating a flow reciprocating in a direction intersecting the introduction direction of the fluid, it is preferable to provide the means in a direction parallel to the introduction direction of the fluid in the flow path, and the pressurizing / depressurizing means is provided in the flow path. When it is a means for generating a flow reciprocating in a direction parallel to the introduction direction of the fluid, it is preferable to provide the means in a direction intersecting the introduction direction of the fluid in the flow path.

Hereinafter, the particle manipulation apparatus of the present invention according to the second aspect will be described in detail with reference to the drawings.
An example of the particle manipulation device of the present invention is shown in FIG. The particle manipulation device 2100 shown in FIG. 16 observes the movement of the flow path structure 210 for introducing the liquid containing the particles, the vibration flow generator (accumulation / depressurization means) 220, and the particles introduced into the flow path structure 210. It is equipped with a particle observation device 230 for the purpose.
The flow path structure 210 was formed by stacking the lower substrate member 211, the side surface member 213, and the upper substrate member 212 in this order from the bottom (FIG. 16). The lower substrate member 211 is a PC (polycarbonate) substrate having a thickness of 0.9 mm and a square of 10 cm, and a concavo-convex portion 11a having a sawtooth-like cross section of 200 μm in width and 5 μm in height extends linearly in a region of 50 mm × 90 mm. A plurality of them were provided (FIG. 17). The upper substrate member 212 is a glass substrate having a thickness of 1 mm and 10 cm square, and has two introduction ports and two φ1 cm through holes 212a (introduction port 212aa and discharge port 212ab) on the center line of the substrate and 1 cm from the end face. It was formed (Fig. 18). A metal electric eyelet is fixed to the through hole 212a with an adhesive for connection with the PTFE tube. By connecting the electric eyelet and the PTFE tube to a syringe pump, a liquid containing particles is introduced and discharged. One discharge port 212ab is sufficient when observing the movement of particles, but it is preferable to add the discharge port 212ab when collecting particles that have moved vertically from the introduction direction of the liquid containing the particles (Fig.). 26). The side surface member 13 is a silicon rubber sheet having a thickness of 1.5 mm and a square of 10 cm, and a region of 50 mm × 90 mm (corresponding to the region provided with the uneven portion 211a extending linearly) is hollowed out to provide an opening 213a. As a result, a planar flow path was formed (FIG. 19). The uneven portion 211a extending linearly is formed in a direction parallel to the introduction direction of the liquid (that is, the direction from the introduction port 212aa to the discharge port 212ab) in the flow path of the liquid containing particles. Further, the above-mentioned flow path structure 210 is a preferable structure for classifying particles having a size of several tens of μm to several hundreds of μm.

The vibration flow generator 220 includes a substrate holder (not shown) for holding the flow path structure 210, a piezoelectric element 221 (manufactured by Shoei System) that can be displaced in the thickness direction of the flow path structure 10, and a piezoelectric element 221. A piezo driver 222 (manufactured by Shoei System, SSL-140-1CH) and a drive signal generator 223 (manufactured by NF circuit block, WF1646B) are provided to generate an oscillating flow (reciprocating flow) by mutating. The piezoelectric element 221 is provided at two locations above the linear uneven portion 211a provided on the flow path structure 210 and in contact with the position 240 1 cm from the substrate end of the flow path structure 210, and has a flow path structure. It can be displaced by 100 μm in the thickness direction of the body 210. Specifically, the piezoelectric element 221a and the output of the piezo driver 222a are connected, the piezoelectric element 221b and the output of the piezo driver 222b are connected, respectively, and the input of the piezo drivers 222a and 222b and the output of the drive signal generator 223 are connected. By connecting, a vibration flow is formed in the liquid contained in the flow path structure 210. To form the vibration flow, the phase of the output waveform of the drive signal generator 223 to the piezo driver 222a and the phase of the output waveform of the piezo driver 222b are shifted by 180 degrees or the signal is inverted to extend the piezoelectric element 221. By alternately driving the contraction, a vibration flow was formed in a direction perpendicular to the introduction direction of the liquid (that is, the direction from the introduction port 212aa to the discharge port 212ab) in the flow path of the liquid containing particles (FIG. 20). The output waveform of the drive signal generator 223 to the piezo driver 222 can be arbitrarily generated, and examples thereof include a sine wave, a triangular wave, a square wave, and a sawtooth wave.

The particle observation device 230 includes a zoom lens 231 (manufactured by Moritex) and a camera 232 (manufactured by Sony CCD camera XCD-V50) capable of observing a region of the flow path structure 210 provided with at least a linearly extending uneven portion 211a. Alternatively, a Tomoei high-speed camera VFC-1000) is provided at the center of the substrate of the flow path structure 210 with a fine movement mechanism (not shown). It is preferable that the camera 232 is switched between a high-speed camera when observing the vibration of particles and a CCD camera when observing at low speed, depending on the situation. By performing binarization processing on each image of one frame of the observation video by the camera 232 (high-speed camera), after detecting the particles introduced into the flow path structure 210, the position of the detected particles in the center of gravity is determined. The movement locus of particles can be measured by measuring and converting the position information into data as a change over time.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these examples.

1. 1. Manufacture of flow path structure As a top substrate member, two through holes with a diameter of 1 mm were machined as a particle introduction port / discharge port 22/23 on a blue plate glass having a thickness of 1.1 mm and a thickness of 10 cm. The position is the position represented by reference numeral 22/23 in the top view of the side member of FIG. 4B. Further, as the lower surface substrate member, the same member as the upper surface substrate member was used except that there was no through hole. As shown in FIG. 4B, a 10 cm × 10 cm, 1 mm thick silicone rubber sheet is cut out so as to form a circular portion having a diameter of 32 mm and a flow path having a width of 5 mm and a length of 42 mm, which are cut out to form a top substrate member and a bottom substrate member. It was arranged between and used as a side member. The upper surface substrate member and the lower surface substrate member and the silicone rubber sheet are joined by the adhesive force of the silicone rubber sheet so as to minimize liquid leakage. The position of the center of the circular hollow portion of the silicone rubber sheet on the upper surface substrate member was set as the arrangement position of the vibrating members 9L and 9R. The sample introduction / discharge port has an eyelet fixed to the through hole of the upper lid substrate member with an adhesive so that a flexible tube can be connected so that the liquid can be introduced and discharged, so that the tube can be connected to the flow path structure. Manufactured.

2. 2. Reciprocating liquid flow generator 100 μm displacement in the substrate thickness direction at a position 1 cm from the edge of the substrate with respect to the position of the substrate holder 11 having a mechanism for holding the flow path structure 2 and the vibrating member arrangement position of the flow path structure 2. Possible piezoelectric elements (manufactured by Shoei System) were arranged as vibrating members 9L and 9R. Two piezodrivers SSL-140-1CH (manufactured by Shoei System) were installed as a vibration member drive power source to form a vibration flow for the two piezoelectric elements. Further, WF1646B (manufactured by NF circuit block) was connected as the waveform signal generator 15 (FIG. 1).

3. 3. Particle observation device A zoom lens (manufactured by Moritex), a CCD camera (XCD-V50 manufactured by SONY) or a high-speed camera (VFC-1000 manufactured by Tomoei) capable of observing the central part of the microchannel structure is provided with a fine movement mechanism in the central part of the substrate. And installed (Fig. 1).

Example 1: Movement of particles having a particle size of 200 μm in ethanol A flow path structure is set in a reciprocating liquid flow generator provided with the above particle observation device, and a liquid containing particles is placed in the flow path structure. Introduced. The introduced particles were Tosoh Toyopearl gel having a particle size of about 200 μm, and were used by substituting and dispersing with 99.5% ethanol (density: 0.7892 g / cm ³ ). In addition, after the particles were introduced, the sample introduction / discharge port was subjected to batch processing by sealing the eyelets fixed with an adhesive in the port and sealing the sample.

As the drive waveforms of the two piezoelectric elements, the drive waveforms were set so that the expansion and contraction of 9L and 9R were reversed as shown in FIG. 3B. The change in flow due to compression / expansion of the circular portion at this time is shown in FIG. 3C for easy consideration. Here, one period of the waveform is expressed as 360 °, and the apex of the triangular wave is expressed as 0 ° as a line-symmetrical vibration on FIG. 3C. From this state, the apex of the triangular wave was changed by a maximum of ± 180 °, and the movement of particles was observed. The frequency of the drive waveform at this time was set to 5 Hz, and the maximum drive voltage of the piezoelectric element was set to 2 V (variable by 100 μm at 5 Vmax) and the extension of the piezoelectric element was set to 40 μm. The movement locus of the particles due to the vibration of the piezoelectric element was measured by automatic measurement of the particle position by image analysis as shown in FIG.

When the piezoelectric element is driven as shown in FIG. 3C in such a state, the reciprocating signal waveform is axisymmetric with respect to the time axis, that is, the apex of the triangular wave is 0 ° (that is, the 0 ° triangular wave in FIG. 3C). Although the 200 μm particle oscillated, no movement in one direction could be observed. On the other hand, in the case of a non-axisymmetric waveform other than line symmetry with respect to the time axis (that is, the apex of the triangular wave is 0 °), that is, in the case of a non-axisymmetric waveform of 60 ° to 150 °, the displacement amount of the reciprocating fluid is also the non-axisymmetric waveform. Become. When the positions of the particles at this time were measured, it was confirmed that the particles moved to the right as shown in FIG. 7A.

Further, it was confirmed that the movement of the particles moved to the left as shown in FIG. 7B when the temperature was set to -60 ° to -150 °, and the larger the deviation of the apex from each triangular wave, the larger the amount of movement of the particles. confirmed. In addition, it was confirmed that the movement direction was reversed and the movement amount was dramatically improved by setting the apex of the triangular wave to ± 180 ° (FIG. 8).

Example 2: Except that Ficoll (density 1.077 g / cm ³ ) was used as a solvent for dispersing the moving particles of particles having a particle size of 200 μm in Ficoll and the driving waveform was changed, the same as in Example 1. The experiment was conducted in the same manner. As the drive waveform of the piezoelectric element, a waveform changed by + 150 ° and −150 ° from the apex of the triangular wave was used. The frequency of the drive waveform at this time is set to 5 Hz, the maximum drive voltage of the piezoelectric element is set to 2 V (fluctuation of 100 μm at 5 Vmax), the extension of the piezoelectric element is set to 40 μm, and the movement trajectory of the particles due to the vibration of the piezoelectric element is an image as shown in FIG. It was measured by automatic measurement of the particle position by analysis.

As a result, as shown in FIG. 8, the positive and negative movement directions did not change, but the movement speed was significantly reduced as compared with the movement speed with ethanol. Further, it was confirmed that when air bubbles (0.0012 g / cm ³ ) were mixed in the solution, the particles moved in the exact opposite direction to the 200 μm particles.

Example 3: In the flow path structure used for moving 20 μm particles of polystyrene beads in a continuous flow system , one particle introduction port is provided in a linear flow path portion having a width of 10 mm, and five particles are located on the opposite side. A silicone rubber sheet was cut out so as to form a particle acquisition port, and a through hole was further formed in the upper surface substrate (FIG. 5). Eyelets were fixed to the through holes with an adhesive so that the tubes could be connected. The flow path was filled with water in advance, the outlet on the opposite side of the silicone tubes attached to the discharge ports 1, 3 and 5 was installed in the sampling container, and the tips of the silicone tubes attached to the discharge ports 2 and 4 were closed. .. While introducing the liquid containing the particles from the particle introduction port, the drive waveforms of the two piezoelectric elements in the photograph were set so that the expansion and contraction of the two piezoelectric elements were reversed. As the drive waveform shape of the piezoelectric element, a sawtooth waveform was given as in the case of FIG. 3C 180 °. The frequency of the drive waveform at this time was set to 5 Hz, and the maximum drive voltage of the piezoelectric element was set to 2 V (variable by 100 μm at 5 Vmax) and the extension of the piezoelectric element was set to 40 μm.

Two types of particle samples, polymer standard particles with a particle size of 20 μm (Duke Standards Cat No. 4220A) and fluorescent particles with a particle size of 1.0 μm (Fluoro-Max (TM) Cat No. G0100), are dispersed in pure water. It was introduced from a syringe pump at 100 μL / min into an introduction port communicating with a flow path communicating the two circular portions.

During stable operation of such a state, the liquid discharged from the discharge ports 1, 3 and 5 was taken for a certain period of time. The liquid volumes from the sorted ports 1, 3 and 5 were 0.65 g, 0.74 g, and 0.60 g, respectively. Then, 50 μL of the collected liquid was collected with uniform stirring and observed under a microscope. When the content of 20 μm particles was counted, the number of particles was 9, 57, and 43 from ports 1, 3 and 5, respectively, and a large amount could be separated from ports 3 and 5, respectively. In addition, a large number of 1 μm particles were detected from any port. In this way, it was confirmed that it is possible to efficiently separate the particles to be separated from the discharge ports in which a plurality of particles are formed.

Example 4: Separation of cancer cells and blood cells As shown in FIG. 10, the flow path used is 10 cm square as a top substrate member and 1.1 mm thick blue plate glass with two 1 mm diameter through holes as sample introduction / discharge ports. processed. Further, as the lower surface substrate member, a member having no through hole is used, and a silicon rubber sheet having a thickness of 1 mm is pressed between the upper surface substrate member and the lower surface substrate member by a piezoelectric element having a diameter of 32 mmφ as shown in FIG. Two circular parts that generate the flow were created. This circular portion is cut out so as to communicate with the through hole of the upper substrate. Further, a linear flow path having a width of 5 mm was formed so as to communicate the two circular portions. The upper surface substrate member substrate, the silicon rubber sheet, and the lower surface substrate member thus prepared were patterned in a comb pattern and joined by the adhesive force of the silicon rubber sheet to form a structure that minimizes liquid leakage. The sample introduction / discharge port has eyelets fixed with adhesive to the through holes of the upper surface substrate member substrate so that flexible tubes can be connected so that liquid can be introduced and discharged so that the tubes can be connected. A particle introduction port 29 was created in the center of the flow path communicating the circular portion and the circular portion of the piezoelectric element compression portion with an injection needle, and a diluted solution of blood cells and cancer cells was introduced.

After filling the mannitol solution from the introduction / discharge port 22/23 in advance, a solution obtained by adding cancer cells (SKBR) to whole blood diluted to 1% with mannitol is introduced from the particle introduction port 29, and all the introduction / discharge ports 22 / 23 was sealed with adhesive tape. As shown in FIG. 1, the center of the expansion / contraction portion 3 of the piezoelectric elements 2L and 2R was aligned with the piezoelectric element contact point (near the circular center) of FIG. As the drive waveforms of the two piezoelectric elements in FIG. 1, the drive waveforms were set so that the expansion and contraction of 2L and 2R were reversed as shown in FIG. 3B. The change in flow due to compression / expansion of the circular portion at this time is shown in FIG. 3C for easy consideration. Here, one period of the waveform is expressed as 360 degrees, and the apex of the triangular wave is expressed as 0 ° as a symmetrical vibration on FIG. 3C. From this state, the apex of the triangular wave was set to + 180 °, and the migration status of blood cells and cancer cells was observed. The frequency of the drive waveform at this time was set to 1 Hz, and the maximum drive voltage of the piezoelectric element was set to 3 V (variable by 100 μm at 5 Vmax) and the extension of the piezoelectric element was set to 60 μm. Since it was difficult to observe the movement of particles with a high-speed camera, the particles were driven for 120 seconds and then stopped to observe the movement status of each particle. As a result, as shown in FIG. 11, it was found that the whole particles moved to the left side from the center of the linear flow path, and in particular, the cancer cells (SKBR) moved to the leftmost side.

Example 5: Device for Manipulating Particles in Batch Units One aspect of the particle manipulation device of the present invention capable of classifying / measuring particles in batch units is shown in FIGS. 14 (principle) and 15 (one aspect of configuration). In the particle separation flow path 35 that communicates the introduction port 32 and the discharge port 34, a certain amount of fluid (sample) is introduced from the sample introduction port 33 and then closed, and at the same time, the vibration flow forming device 38 causes the discharge port to be discharged from the introduction port 32. By forming the oscillating flow 36 parallel to the flow direction (mainstream direction) to 34, the particles contained in the sample introduced from the sample introduction port 33 are separated toward the discharge port 34 based on the particle size. At this time, if an optical detection device 44 such as a UV detector is connected to the discharge port 34 side, the separation peak 45 can be detected, and the particle size distribution can be measured in the same manner as in chromatography. If a light scattering detector is used as the optical detection device 44, the particle size can be measured.

Example 6
(1) The particle manipulation device 2100 of the present invention shown below was produced.
(1-1) Lower substrate member 211
It is a PC (polycarbonate) substrate having a thickness of 0.9 mm and a square of 10 cm, and a concavo-convex portion 211a having a sawtooth wavy cross section with a width of 200 μm and a height of 20 μm and extending linearly is provided in a region of 50 mm × 90 mm (FIG. 17). ).
(1-2) Upper substrate member 212
It is the same as the particle manipulation device 2100 of the present invention shown in FIG.
(1-3) Side member 213
A silicon rubber sheet having a thickness of 1.5 mm and a square of 10 cm, of which a hexagonal region of 70 mm × 90 mm was hollowed out and an opening 213a was provided to form a planar flow path (FIG. 21).
(1-4) Vibration flow generator 220
By inverting the output signal to the piezo driver 222a of the drive signal generator 223 and the output signal to the piezo driver 222b and alternately driving the expansion / contraction of the piezoelectric element 221, the above-mentioned in the flow path of the liquid containing particles. It is the same as the particle manipulation device 2100 of the present invention shown in FIG. 16, except that the vibration flow is generated in the direction perpendicular to the liquid introduction direction (that is, the direction from the introduction port 212aa to the discharge port 212ab).
(1-5) Particle observation device 230
It is the same as the particle manipulation device 2100 of the present invention shown in FIG.
(2) From the introduction port 212aa of the particle manipulation device 2100 of the present invention produced in (1), a 99.5% ethanol solution containing Toyopearl (manufactured by Tosoh) particles having a diameter of 200 μm is injected through a PTFE tube using a syringe. It was introduced until the opening 213a was filled with the solution.
(3) The sawtooth wave shown in the patterns (a) and (b) of FIG. 22 was output from the drive signal generator 223 to move the Toyopearl particles.
As a result, when the sawtooth wave of the pattern (a) was output, the particles moved to the left, and when the sawtooth wave of the pattern (b) was output, the particles moved to the right.

Example 7
(1) A device similar to the particle manipulation device 2100 manufactured in Example 6 (1), except that the concavo-convex portion 211a provided on the lower substrate member 211 has a sawtooth-like linearly extending uneven portion 211a having a width of 200 μm and a height of 5 μm. Was produced.
(2) The same method as described in Examples 6 (2) to (3) was used except that a 99.5% ethanol solution containing φ200 μm and φ80 μm Toyopearl (manufactured by Tosoh) particles was used as the solution containing the particles. The Toyopearl particles were moved.
(3) Introduced into the flow path structure 210 by subjecting each frame of the observation video obtained by the particle observation device 230 (camera 232 is a high-speed camera VFC-1000 manufactured by Toei) to a binarization process. After detecting the particles, the position of the detected particles in the center of gravity was measured, and the movement locus of the Toyopearl particles was measured by converting the position information into data as a temporal change.
The results are shown in FIG. It can be seen that the moving speed of the toyopearl particles having a large particle size of φ200 μm is relatively high, and the moving speed of the toyopearl particles having a small particle size of φ80 μm is relatively slow. Further, it can be seen that when the particle vibration width exceeds the interval (200 μm) of the uneven portions 211a extending in a sawtooth shape, the moving speed of the particles becomes dramatically faster.

Example 8
(1) A device similar to the particle manipulation device 2100 manufactured in Example 7 was manufactured except that the uneven portion 211a provided on the lower substrate member 211 having a sawtooth-like linearly extending uneven portion 211a had a width of 50 μm and a height of 1 μm. ..
(2) The same as in Examples 7 (2) and (3) except that a 99.5% ethanol solution containing φ200 μm, φ80 μm and φ40 μm Toyopearl (manufactured by Tosoh) particles was used as the solution containing the particles. The Toyopearl particles were moved by the method.
(3) The movement locus of the Toyopearl particles was measured by the same method as described in Example 7 (3).
The results are shown in FIG. It can be seen that the moving speed of the Toyopearl particles is relatively faster in descending order of particle size (φ200 μm> φ80 μm> φ40 μm). Further, it can be seen that when the particle vibration width exceeds the interval (50 μm) of 211a extending in a straight line like a sawtooth, the moving speed of the particles becomes dramatically faster.
From the results shown in FIGS. 23 and 24, in the particle manipulation device of the present invention of the second aspect, the direction perpendicular to the introduction direction of the liquid containing the particles (that is, the direction from the introduction port 212aa to the discharge port 212ab). It is suggested that classification by particle size (diameter) is possible by providing a particle discharge port.

Comparative Example 1
(1) Using the particle manipulation device 2100 produced in Example 6, a solution containing particles was introduced in the same manner as in Example 7 (2) until the opening 213a was filled with the solution.
(2) From the drive signal generator 223, the sine waves shown in the patterns (a) and (b) of FIG. 25 were output, and the Toyopearl particles were moved.
As a result, when a sine wave of any pattern was output, the Toyopearl particles only vibrated, and no physical movement could be confirmed. The same result was obtained when the sine wave was changed to a triangular wave or a square wave. From this result, it can be seen that in the particle manipulation device of the present invention, if the waveform indicating the amount of displacement of the liquid in the vibration flow is only a point-symmetrical waveform or only a line-symmetrical waveform, the particles do not move.

1 Reciprocating liquid flow generator 2 Flow path structure 3 Liquid feed pump 4 Tank 5 Liquid feed tube 6 Drainage / fluid acquisition tube 7 Camera 8 Zoom lens 9L Vibration member 9R Vibration member 10 Vibration member holder 11 Board holder 12L Vibration member Drive power supply 12R Vibration member drive power supply 13L Vibration member drive signal output 13R Vibration member drive signal output 14L Vibration member drive source signal input 14R Vibration member drive source signal input 15 Wave signal generator 16L 12L waveform output 16R 12R waveform output 17 100 μm Enlarged view of the stretchable part of the expandable piezoelectric element 18 Top substrate 19 Side member 20 Bottom substrate 21 Vibration member adhesion point 22 Fluid inlet 23 Fluid discharge port 24 Flow path 25 Particle acquisition port 26 Liquid flow to the right 27 Left direction Liquid flow to 28 Particles 29 Particle introduction port 30 Diaphragm flow path 31 Particle separation flow path 32 Liquid introduction port 33 Sample introduction port (sample injector)
34 Solution Discharge Port 35 Particle Separation Channel 36 Vibration Flow 37 Separation Band Image 38 Vibration Flow Forming Device 39 Solution Leather Bar 40 Liquid Feed Pump 41 Vibration Flow Forming Device 42 Sample Injector 43 Separation Flow Path 44 Optical Detector 45 Separation Peak 46 Discharge Port 47 Diaphragm introduction / discharge port 48 Particle separation flow path introduction / discharge port 2100 Particle manipulation device 210 Flow path structure 211 Lower board member 211a Concavo-convex portion extending linearly 211ab Discharge port 212 Upper board member 212aa Introduction port 212ab Discharge port 213 Side member 213a Opening 220 Vibration flow generator (accumulation / depressurization means)
221 Piezoelectric element 222 Piezodriver 223 Drive signal generator 230 Particle observation device 231 Zoom lens 232 Camera 240 Piezoelectric element contact point in flow path structure 2200 Vibration flow (reciprocating flow)

Claims (15)

And one or more fluid inlets, one or fluid flow path comprising particles and a plurality of fluid outlets, and periodically reciprocating flow to the main flow which is continuously fed to intertwine direction A particle separation detection device including one or more heating / depressurizing means to be generated and a detection device capable of detecting the passage of particles connected to the fluid discharge port, and a waveform showing the displacement amount of the fluid in the reciprocating flow. The particle separation detection device having a non-linear symmetric waveform. The particle separation detection device according to claim 1, wherein the flow path is formed between the upper surface substrate member, the lower surface substrate member, and the side surface member. The particle separation detection device according to claim 2, wherein the pressurizing / depressurizing means is arranged on one or a plurality of members selected from the group consisting of the upper surface substrate member, the lower surface substrate member, and the side surface member. A particle manipulation device comprising a flow path of a fluid containing particles and one or more pressurizing / depressurizing means for generating reciprocating flow.
The pressurizing / depressurizing means is a means for generating a reciprocating flow in a direction parallel to or intersecting the introduction direction of the fluid in the flow path.
The waveform indicating the displacement amount of the fluid in the reciprocating flow has a non-axisymmetric waveform.
The particle manipulation device, which is provided with one or more uneven portions in the flow path. The particle manipulation device according to claim 4, wherein the flow path is formed between the upper substrate member, the lower substrate member, and the side surface member. The particle manipulation device according to claim 5, wherein the uneven portion is arranged on either one or both of the upper substrate member and the lower substrate member, and extends in a direction parallel to the introduction direction of the fluid in the flow path. The particle manipulation device according to claim 6, wherein the uneven portion provided in the flow path extends linearly. The particle manipulation device according to claim 7, wherein the cross section of the uneven portion extending in a straight line has a non-axisymmetric shape. The particle manipulation device according to any one of claims 4 to 8, wherein the pressurizing / depressurizing means is a means for generating a flow reciprocating in a direction perpendicular to the introduction direction of the fluid in the flow path. The particle according to any one of claims 4 to 9, wherein a plurality of particle discharge ports are provided in the flow path of the fluid containing the particles, and the particle discharge port is provided in the direction perpendicular to the introduction direction of the fluid containing the particles. Operating device. The particle manipulation device according to claim 10, wherein particles having different diameters are discharged from each particle discharge port. A method for classifying particles contained in a fluid using the particle manipulation device according to any one of claims 4 to 11. A particle manipulation device comprising a flow path of a fluid containing particles and one or more pressurizing / depressurizing means for generating reciprocating flow.
The pressurizing / depressurizing means is a means for generating a flow reciprocating in a direction parallel to the introduction direction of the fluid in the flow path.
The waveform indicating the displacement amount of the fluid in the reciprocating flow has a non-axisymmetric waveform.
The particle manipulation device, which is provided with one or more uneven portions in the flow path. The particle manipulation device according to claim 13, wherein the flow path is formed between the upper substrate member, the lower substrate member, and the side surface member. The particle manipulation device according to claim 14, wherein the uneven portion is arranged on either one or both of the upper substrate member and the lower substrate member, and extends in a direction intersecting the introduction direction of the fluid in the flow path.