JP2011104487A - Apparatus for treating fine particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a low cost and simple-structured apparatus for treating fine particles, requiring least expense, time and skill in its maintenance and allowing an operator to carry out a highly accurate treatment of fine particles without requiring a specific skill. <P>SOLUTION: The apparatus for treating fine particles including a storage section that stores a dispersion of fine particles, an electrode base plate whereon a pair of electrodes are disposed, and an AC power source connected to the pair of electrodes, is characterized in that a part of the storage section is constituted of an insulating material and comprises a plurality of through-holes enabling the suspension to contact each of the electrodes. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a particle manipulation device that guides and captures fine particles at a specific position.

  Non-contact, non-destructive capture, retention, selection, transfer of inorganic material-based fine particles, organic material-based fine particles, biological sample-based fine particles, etc., without damaging the physical shape and physical properties of each fine particle For example, a method for manipulating fine particles by light, a device for manipulating fine particles by light, and the like have been used as a method and an apparatus for separating and collecting (see, for example, Patent Document 1. In this specification, fine particles are non-contact and non-destructive. Capture, hold, select, move, separate collection, etc. in the above are sometimes simply expressed as “operating”.) This operation method is generally called an optical tweezer method or an optical trap method, and is also called a laser trapping method because a laser is mainly used as a light source. Specifically, the laser light from the light source is condensed in a conical shape by a condensing optical system, and irradiated to the vicinity of the fine particles in the medium, so that the fine particles are captured and held using the radiation pressure of the light generated in the fine particles. And move it.

  However, the optical trap method requires a laser light source and an optical system for condensing the light source, and requires precise control thereof, so that the apparatus becomes expensive, precise and large-scale, and handling is complicated. Therefore, there is a problem that the operator is forced to have a certain level of skill. In addition, the maintenance of the optical system and the light source including the optical axis adjustment requires time, cost, and skill, and the management cost increases accordingly. Furthermore, the optical trap method has a problem that a large amount of fine particles cannot be manipulated at a time because the fine particles must basically be captured one by one while visually confirming with a microscope or the like.

  Microparticle manipulation methods and manipulation devices such as the optical trap method are regarded as basic techniques for cell fusion by capturing different biological sample microparticles such as cells and fusing them into one hybrid cell. (For example, refer to Patent Document 4). However, as described above, the apparatus becomes expensive, precise and large-scale, and the handling becomes complicated, and the operator is required to have a certain level of skill. In addition, because the skill is required and the management cost increases accordingly, it is necessary to capture each particle while visually confirming them one by one with a microscope, etc., so that a large amount of particles cannot be manipulated at one time. Further improvement is necessary to improve

  As another known technique related to the manipulation of biological sample-based microparticles such as cells, for example, as disclosed in Patent Document 2, a pair of electrodes made of a conductive member arranged to face the fusion region of the cell fusion chamber, A cell fusion chamber comprising an insulator having a through hole disposed between the pair of electrodes and penetrating in the direction of the pair of electrodes has been reported.

  Patent Document 2 will be specifically described. FIG. 1 is a conceptual diagram showing a cross-sectional view of the cell fusion chamber of Patent Document 2. In FIG. 1, electrodes (2) made of conductive members are arranged on both sides of a fusion region (1) of a cell fusion chamber made of a resin material, for example, and these electrodes are provided outside via conductive wires (3). Connected to the power source (4). The power supply provided outside is an AC power supply (5) that outputs a high-frequency AC voltage with an electric field strength of about 400 V / cm to 700 V / cm and a frequency of about 1 MHz, and a pulse voltage of about 7 kV / cm and a pulse width of about 50 μsec. And a switch (7) for switching the electrical connection between the electrode and the AC power source or the DC pulse power source. Here, a general sine wave waveform is usually used as the AC voltage output from the AC power supply. The cell fusion chamber is divided into two spaces by an insulator (8) made of an electrically insulating material such as silicone resin. Here, the insulator is provided with a through hole (9) having a minimum diameter of 1 μm to several tens of μm. Cell A (10) and cell B (11) are each placed in a suspension in the fusion region of the cell fusion chamber.

  The operation of the apparatus described in Patent Document 2 will be specifically described with reference to FIGS. First, the changeover switch (7) of the power source (4) is connected to an AC power source (5) that outputs a high-frequency voltage having an electric field strength of about 400 V / cm to 700 V / cm and a frequency of 1 MHz. In this state, the electric lines of force (12) are concentrated in the through hole (9) as shown in FIG. The cells A (10) and B (11) receive a dielectrophoretic force due to the electric lines of force (12) concentrated here, and are captured near the center of the through hole (9) as shown in FIG. Here, cell A (10) and cell B (11) meet and come into contact. Next, the selector switch (7) of the power source (4) is switched to the DC pulse power source (6). In the cells A (10) and B (11) placed in the state shown in FIG. 3, the reversible destruction of the cell membrane occurs at the contact point of the cells A (10) and B (11) due to the pulse voltage. Fusion occurs as shown in FIG.

  The device of Patent Document 2 is intended to fuse cell A and cell B in the through-hole, and therefore handles two cells as a pair. Therefore, when trying to obtain a plurality of fused cells (51) at once by the apparatus described in Patent Document 2, it is necessary to capture a plurality of pairs of two through holes on the insulator as a pair of two cells. However, when an AC power source is connected and cells are captured in the through-holes, a plurality of cells are captured in a certain through-hole, while a through-hole in which no cells are captured is generated. There is a problem that cell manipulation (capturing in uneven through-holes) occurs. Therefore, by arranging the through-holes in an array (by arranging the through-holes at equal intervals with any adjacent through-holes, or by arranging the through-holes in a lattice shape with the same length and width) Attempts have also been made to uniformize the cell operation (capture in the through-hole) by uniforming the acting electric field lines.

  Patent Document 3 discloses a method of capturing cells one by one in through holes arranged in an array (see Patent Document 3). This method penetrates a suspension containing a plurality of cells into a through-hole whose inside diameter and depth of the through-hole (microwell) are 1 to 2 times the particle size of the cell (subject lymphocyte). In addition to covering the hole, the process of waiting for the cells to sink into the through hole and the washing process of washing away cells other than the cells that have settled in the through hole are repeated to capture one cell in one through hole. To do. However, in the method described in Patent Document 3, the time for waiting for the cells to sink due to gravity is as long as about 5 minutes, the process of waiting for the cells to sink in the through holes, and the cells that have not entered the through holes are washed away. There were problems that the operation was cumbersome and complicated to repeat the washing process, and it was difficult to perform rapid processing, and that the cells that entered the through hole could be lost in the process of washing the cells that did not enter the through hole . In particular, after treating (selecting) cells for any specificity, it is a great loss to wash away cells that have not entered the through-hole.

JP 2001-290083 A Japanese Patent Publication No. 7-4218 Japanese Patent No. 3723882 JP-A-7-31455

  The present invention has been proposed in view of such a conventional situation, and an object thereof is an inexpensive apparatus having a simple configuration, and does not require time, cost, and skill for maintenance and management. An object of the present invention is to provide an operation device for fine particles. Another object of the present invention is to provide an apparatus that can process fine particles with high accuracy without requiring any special skill in use. Furthermore, the objective of this invention is providing the processing apparatus which can process a lot of fine particles at once.

  The present invention, which has been made to achieve the above object, comprises a housing part for housing a fine particle suspension, an electrode substrate on which a pair of electrodes are arranged, and an AC power source connected to the electrodes. The part is a fine particle manipulating device characterized in that it is made of an insulating material and has a through-hole that allows the suspension to contact each electrode. Hereinafter, the present invention will be described in detail with reference to the drawings.

  The fine particles that can be handled in the present invention are not particularly limited as long as they are electrically capacitive fine particles. For example, inorganic material-based fine particles such as silica, zirconia or nickel oxide, organic material-based fine particles such as polystyrene, Biological sample microparticles such as antibody-producing cells and myeloma cells can be exemplified. The size of the particles is not limited as long as it is a size that can move in the suspension by dielectrophoresis, which will be described later. The fine particle suspension used in the apparatus may be a suspension in which the fine particles as described above can move by dielectrophoresis described later. For example, when the fine particles are inorganic material-based fine particles, an aqueous solution in which zirconia particles having a particle diameter of several μm to several hundred μm are suspended, and when the fine particles are organic material-based fine particles, the particle diameter is from several μm to several μm. An aqueous solution in which polystyrene particles of about 100 μm are suspended is suspended in the case where the microparticles are biological sample microparticles, in which antibody-producing cells (size of about 5 μm) and mouse myeloma cells (size of about 10 μm) are suspended. A mannitol aqueous solution (mannitol concentration: about 300 mM) can be exemplified.

  The suspension in which the fine particles are suspended is accommodated in the accommodating portion. The accommodating portion has a through hole that allows a part of the accommodating portion to be made of an insulating material and allows the suspension to contact an electrode disposed on an electrode substrate constituting the device of the present invention. As long as this condition is satisfied, the storage portion is not particularly limited in size and shape, and is not limited to the storage portion in which the suspension is stored in a sealed state. For example, the storage portion may be a liquid reservoir without an upper lid, for example, separately. It may be a part of the flow path connected to the constructed cell extraction device. As an example without the upper lid, for example, in the configuration of FIG. 5, the configuration of FIG. 8 with the upper lid 14 omitted may be illustrated (FIG. 9 is a diagram showing a BB ′ cross section of the configuration of FIG. 8). . The upper lid prevents the water in the suspension containing the fine particles contained in the storage portion from evaporating, and in the apparatus as shown in FIG. 5, the suspension containing the fine particles is stably supplied to the apparatus. Has a role.

  Further, the housing portion is connected to the electrode substrate so as to block a through-hole formed in a part of the housing portion with an electrode disposed on the electrode substrate (see FIG. 6). Here, the part having a through hole is made of an insulating material because, when an AC voltage is applied to the electrode, the lines of electric force are concentrated on an arbitrary through hole, and the fine particles are moved to the through hole. It is for letting it capture. Examples of the insulator material that can be employed in the present invention include glass, ceramic, resin, and the like. However, considering ease of processing when forming the through-hole, resin or the like can be exemplified as a preferable insulator.

  Further, the insulator material forming a part of the accommodating portion is preferably an insulator having affinity with the fine particles because the fine particles are attracted and captured by the through holes formed therein. The insulator material having affinity for the fine particles is preferably a hydrophilic insulator when the fine particles are hydrophilic, and a hydrophobic insulator when the fine particles are hydrophobic. A measure of affinity is generally indicated by the contact angle between the droplet formed when a liquid having affinity close to the fine particles is dropped on the surface of the insulator and the surface of the insulator (contact). The smaller the angle, the higher the affinity between the liquid and the surface of the insulator, and the larger the contact angle, the lower the affinity between the liquid and the surface of the insulator). Insulators with relatively high hydrophilicity include glass and titanium oxide, and insulators with relatively high hydrophobicity include resins such as polystyrene, polyimide, and Teflon (registered trademark). The use of these materials as insulators can be exemplified depending on the hydrophobicity.

Even in the case where an insulator having essentially low affinity with the fine particles must be used, the affinity with the fine particles can be increased by modifying the surface of the insulator. As a method of hydrophilizing a hydrophobic insulator such as a resin, a known method such as plasma treatment, chemical modification, modification by protein physical adsorption, or any combination of these methods may be used. Good. Here, the plasma treatment of the insulator surface refers to the surface of the insulator by irradiating the insulator surface with an electrically neutral ionized gas (plasma) in which active species such as electrons, ions, and radicals are present. In this process, the surface of the insulator is modified by removing organic contaminants and changing the chemical bonding state. Plasma treatment includes plasma surface treatment using a non-polymerizable gas (Ar, O _2, etc.) and plasma polymerization in which an insulator surface is coated with a polymer thin film using an organic monomer. Plasma surface treatment includes formation of a surface cross-linked layer with a non-reactive gas such as Ar, introduction of a functional group with a reactive gas such as O _2, etc., and —COOH or —CO is introduced by oxygen plasma treatment, and an insulator It is possible to increase the affinity with hydrophilic fine particles by improving the hydrophilicity of the surface. In addition, hydrophilicity by chemical modification of the insulator surface here means hydrophilicity by bonding a derivative having a hydrophilic group such as a hydroxyl group, a carboxyl group, an amino group or a sulfone group or a silane coupling agent to the surface of the insulator. It is a method to do. A silane coupling agent is a compound composed of an organic substance and silicon. In the molecule, hydrophilic reactive groups (hydroxyl group, carboxyl group, amino group, sulfone group, etc.) and hydrophobic reactive groups (vinyl group, methyl group) Group, ethyl group, propyl group, etc.) having two or more different reactive groups. For this reason, when a hydrophobic insulator is immersed in a dilute solution of a silane coupling agent, the reactive group showing the hydrophobicity of the silane coupling agent is chemically bonded to the surface of the hydrophobic material, thereby showing hydrophilicity. Since the reactive group covers the surface, the surface of the hydrophobic material can be easily hydrophilized uniformly. In addition, here, in the case of modification by physical adsorption of protein, the protein is physically adsorbed by immersing the insulator in a protein-containing solution such as BSA (bovine serum albumin) for several minutes to several hours. The surface can be hydrophilized.

  As a method of hydrophobizing a hydrophilic insulator such as glass, there is a method by chemical modification in which a silane coupling agent is bonded to the hydrophilic insulator surface. A silane coupling agent is a compound composed of an organic substance and silicon. In the molecule, hydrophilic reactive groups (hydroxyl group, carboxyl group, amino group, sulfone group, etc.) and hydrophobic reactive groups (vinyl group, methyl group) Group, ethyl group, propyl group, etc.) having two or more different reactive groups. Therefore, if a hydrophilic insulator is immersed in a dilute solution of a silane coupling agent, the reactive group showing the hydrophilicity of the silane coupling agent is chemically bonded to the surface of the hydrophilic insulator and exhibits hydrophobicity. Since the reactive group covers the surface, it is possible to uniformly hydrophobize the surface of the insulator.

  In addition, as a hydrophilicity or hydrophobicity evaluation method, the following general methods can be used. That is, pure water is dropped on the surface of the insulator, and the hydrophilicity and hydrophobicity of the surface of the insulator are measured by measuring the contact angle between the droplet formed on the surface of the insulator and the surface of the insulator. Evaluate. Since there is no strict definition of hydrophilicity and hydrophobicity, in the present invention, hydrophilicity is defined as the contact angle of 50 ° or less, preferably 40 ° or less, and hydrophobicity is defined as 50 ° or less. It is defined as greater than, preferably greater than 60 °. Furthermore, the measurement of the contact angle uses the θ / 2 method for calculating the contact angle from the angle of the straight line connecting the left and right end points and the vertex of the droplet dropped on the substrate with respect to the solid surface.

  In order to form the through hole in the insulator, various methods according to the type of the insulator can be employed. For example, in order to form a through hole in the resin, a known method such as a method of irradiating a laser or a method of forming a housing portion using a mold having a pin for forming a through hole can be used. In the case of using a photocurable resin or the like, the through hole can be formed by general photolithography (exposure) and etching (development) using an exposure photomask on which a pattern corresponding to the through hole is drawn.

  In the fine particle manipulating apparatus of the present invention illustrated in FIG. 5, the main body 13 of the apparatus has an accommodating portion composed of an upper lid 14, a spacer 16, a flat insulator 8 having a through hole, and an electrode substrate 15 on which electrodes are arranged. It consists of and. For example, if the portion having the through hole in the housing portion has a box shape capable of sealing the housing portion, the specific gravity of the fine particles is not more than the specific gravity of the suspension containing the fine particles, and the fine particles float upward. In the case, it can be the upper surface. However, considering that gravity can also be used for the operation of fine particles, and that it is easier to carry out the collection operation from the top when separating and collecting fine particles that have been moved and trapped after the operation, etc. It is particularly preferable that the part is a lower part of the accommodated part, and at the same time, a part (14 in FIG. 5) that closes the upper part is removable. Even if the specific gravity of the fine particles is small, it is relatively easy to make the specific gravity of the suspension below that.

  The housing portion can be entirely made of an insulating material. For example, as shown in FIG. 5, the portion (the portion 8 having a through hole) in which the housing portion is made of an insulating material, and other parts It is also possible to divide into parts (16 and 14). For example, a frame or spacer 16 is formed of a material that is easy to process, and a resin flat plate 8 provided with a through hole so that the suspension does not leak is used as a bottom surface to be combined with the frame or spacer to form a housing portion. Can be illustrated.

  The case where the accommodating portion is constituted by the upper lid 14, the spacer 16, and the insulator 8 as shown in FIG. The spacer 16 is for securing a space for actually holding a suspension of fine particles. For example, an insulator such as glass, ceramic, or resin may be used as a material, and the spacer may be electrodes a and b. As long as they are not electrically connected to both (described later), a conductor such as metal may be used as a material. In the example of FIG. 5, the introduction channel and the introduction port 19 communicating with the channel, the discharge channel for discharging the suspension, and the discharge port 20 communicating with the channel are provided in the spacer, and the suspension for the fine particle manipulating device is provided. Suspension can be supplied and discharged quickly. Although there are no particular restrictions on the size and shape of the spacer, it is preferable that the size and shape match a pair of electrodes arranged on the electrode substrate 15. The space inside the spacer and the thickness may be determined in relation to the amount of suspension to be operated, and there is no particular limitation, but usually there should be a capacity to put a microparticle suspension of several μL to several mL. For example, if the spacer size is about 40 mm long x 40 mm wide, the space inside the spacer may be about 20 mm long x 20 mm wide, and the spacer thickness should be about 0.5 to 2.0 mm. That's fine.

  The insulator 8 is configured with an array of through-holes 9 positioned immediately above the pair of electrodes. Strictly speaking, the array shape means that the vertical and horizontal intervals of the through holes are arranged at equal intervals. However, in the present invention, the through holes are linear only in the vertical direction or only in the horizontal direction. In addition, the case where they are arranged at equal intervals is also expressed as an array. By arranging the through holes in an array like this, the electric field generated by the AC voltage applied between the electrodes is generated almost evenly in all the through holes, and uniform operation is realized in the apparatus of the present invention. It is effective.

  A pair of electrodes for applying an alternating voltage is disposed on the electrode substrate connected to the housing portion so as to close the through hole. The electrode substrate is not particularly limited except that an insulator is used to form a pair of electrodes on the electrode substrate. For example, glass, ceramic, resin, etc. can be illustrated as an example of the insulator used as a material.

  The material of the electrode is not particularly limited as long as it is a conductive member and is a chemically stable member, such as metals such as platinum, gold, and copper, alloys such as stainless steel, and ITO (Indium Tin Oxide). A transparent conductive material or the like can also be used. In particular, when the electrode substrate is made of transparent glass or the like for the purpose of monitoring the behavior of the fine particles in the container when the suspension is supplied to the apparatus of the present invention, ITO is used for its transparency and composition. This is a particularly preferable material in terms of film properties. A pair of electrodes 31 composed of electrodes 29 and 30 arranged on the electrode substrate is connected to an AC power source 4 (hereinafter, in this specification, one of the pair of electrodes is an electrode a for convenience and the other is an electrode. b). Electrodes a and b (FIG. 5 shows an example in which four electrodes a and four electrodes b are arranged) correspond to all the through holes of the housing portion. Of course, there may be a plurality of independent electrodes a and b corresponding to the number of through-holes (a + b = the number of through-holes), but usually a large number to enable manipulation of a large number of fine particles simultaneously. It is preferable to use an accommodating portion in which a through hole is formed. Therefore, if a plurality of independent electrodes a and b are arranged so as to correspond to each of them, the configuration of the apparatus becomes complicated, and the maintenance of each electrode is also possible. It becomes complicated. Therefore, in the present invention, as shown in FIG. 5, the pair of electrodes is composed of two parallel electrodes extending from each of two opposing sides toward the other side, or two opposing sides. It is preferable to comprise three or more parallel and equidistant electrodes extending from each of them toward the other side. FIG. 5 shows an example in which four electrodes are arranged in a comb shape as electrodes a constituting one of the pair of electrodes and four electrodes b constituting the pair other in accordance with this embodiment.

  FIG. 6 is a schematic view showing a cross-sectional view taken along the line A-A ′ of the fine particle manipulating apparatus shown in FIG. 5. As a means for bonding the accommodating portion composed of the upper lid 14, the spacer 16, and the insulator 8 and the electrode substrate 15 on which the pair of comb-shaped electrodes 31 (electrodes a 29 and b 30) are arranged, each is bonded with an adhesive. A method, a method of heating and fusing under pressure, a method using a surface-adhesive resin such as PDMS (poly-dimethylsiloxane) or a silicon sheet as a spacer, and a method of bonding by pressure bonding Can be illustrated.

  FIG. 7 is a view showing a state in which the relationship between the arrangement of the electrode substrate 15, the pair of electrodes 31 and the through holes 9 of the fine particle manipulating apparatus shown in FIG. As shown in FIG. 7, in the fine particle manipulating apparatus of the present invention, either the electrode a29 or the electrode b30 of the comb-like electrode 31 is disposed directly below the through-hole 9 penetrating in the vertical direction, and the column or row of the through-holes is arranged. Each is characterized in that the electrodes a29 and b30 are alternately arranged. In such a configuration, the width of the electrode formed immediately below the through hole is not particularly limited as long as it does not overlap with the adjacent electrode, but the through hole is formed so that the lines of electric force are evenly concentrated in the through hole. It is preferable that the diameter be equal to or greater than the diameter. Further, the position of the electrode seen from above the through hole is not particularly limited, but the central axis 32 and the electrode of the through hole are formed as shown in FIG. 7 so that the electric lines of force are evenly concentrated in the through hole. It is preferable to arrange so that the central axes 35 of the conductive members to be overlapped.

  FIG. 10 shows a conventional fine particle manipulating apparatus, and FIG. 11 shows a CC ′ cross section of the apparatus of FIG. 10, which is described for explaining the superiority of the fine particle manipulating apparatus of the present invention. . In the apparatus of FIG. 10, one of the pair of electrodes is disposed on the lower surface of the upper electrode substrate 47 that also functions as an upper lid, and the other of the pair of electrodes is disposed on the upper surface of the lower electrode substrate 48. The pair of electrodes is configured so as to sandwich a housing 16 composed of a spacer 16, an upper lid (upper electrode substrate 48), and a flat plate insulator 8 having a through hole. In the apparatus shown in FIG. 10, since the pair of electrodes sandwich the accommodating portion, the upper electrode substrate 47 functioning as an upper lid cannot be easily removed, and after moving and capturing the fine particles in the through holes, It is difficult to collect arbitrary fine particles captured from the through-hole using a fine particle collecting means such as a micropipette. On the other hand, the apparatus of the present invention has a structure in which a through hole is formed in a part of the accommodating portion and is closed with an electrode substrate in which a pair of electrodes are arranged on the same surface, and thus the upper lid itself is omitted. Even it is possible. Even when the configuration illustrated in FIG. 5 is used for the purpose of preventing the evaporation of the suspension, the apparatus of the present invention makes all the pair of electrodes (electrode a29 and electrode b30 in FIG. 5) comb-like or the like. It is possible to remove the upper lid because it is placed on a single electrode substrate, and above all, while applying an AC voltage to the electrode to cause the dielectrophoretic force to act on the fine particles, Any of the captured fine particles can be easily collected with a micropipette or the like.

  In the apparatus of the present invention, in addition to the above, since all the pair of electrodes are installed on one electrode substrate, the distance between the pair of electrodes is several μm to several tens μm such as the particle diameter of the fine particles. It is possible to make it minute. As a result, even if the alternating voltage applied between the pair of electrodes is low, it is possible to generate a large electric field between the electrodes due to the minute distance between the electrodes and to apply a large dielectrophoretic force to the fine particles. For example, in the conventional fine particle manipulating apparatus shown in FIG. 10, a pair of electrodes are disposed on the lower surface of the upper electrode substrate 47 and the upper surface of the lower electrode substrate 48, and the spacer 16 is sandwiched between them to serve as an accommodating portion. The distance is limited by the thickness of the spacer. Generally, if the spacer thickness is less than 1 mm, it is difficult to introduce a suspension of fine particles into the accommodating portion. Therefore, the distance between the electrode substrates is less than 1 mm. I can't do it. Here, since the electric field is a value obtained by dividing the voltage applied between the electrodes by the distance between the electrodes, the device of the present invention (for example, the device shown in FIG. 5) that can make the separation distance between the electrodes several μm to several tens μm. ), It is possible to obtain an electric field several tens of times stronger if the same voltage is applied as compared with the conventional apparatus shown in FIG. 10 where the same distance cannot be less than 1 mm. If it is obtained, a voltage of several tenths is sufficient. As a result, it is possible to provide a device that enables more effective particle manipulation than conventional devices even when a small and inexpensive AC power supply is used.

  The AC power supply 4 is connected to the electrode a29 and the electrode b30, which are a pair of electrodes disposed on the electrode substrate 15 of the fine particle manipulation device, through the conductive wire 3. The AC power supply 4 is not particularly limited as long as it can apply an AC voltage between the electrodes a and b sufficient to move and trap the fine particles into the through hole and generate an electric field that causes the fine particles to be removed from the through hole. Specifically, for example, a power source that can apply an AC voltage having a peak voltage of about 1 V to 20 V and a frequency of about 100 kHz to 3 MHz such as a sine wave, a rectangular wave, a triangular wave, a trapezoidal wave, etc. It is particularly preferable to apply a waveform alternating voltage between the electrodes a29 and b30 so that only one fine particle can be captured in one through-hole. A rectangular wave is preferably used as the AC voltage having such a waveform. As shown in FIGS. 12 to 15, when the waveform of the AC voltage is a rectangular wave (FIG. 15) compared to a sine wave (FIG. 12), a triangular wave (FIG. 13), and a trapezoidal wave (FIG. 14). Since the peak voltage 36 set instantaneously is reached, the fine particles can be quickly moved toward the through hole, and the probability of entering the through hole so that two or more fine particles overlap each other can be reduced (one The probability that only one fine particle can be trapped in the through hole is increased). The fine particles can be regarded as electrical capacitors. However, while the peak voltage of the rectangular wave does not change, it is difficult for current to flow through the fine particles trapped in the through-holes, and it is difficult to generate electric lines of force. The dielectrophoretic force is less likely to be generated in the through-hole that has captured Therefore, once a fine particle is trapped in a through hole, the probability that another fine particle is trapped in that through hole is low, and instead a through hole (capturing a fine particle) in which electric force lines are generated and a dielectrophoretic force is generated. This is because the fine particles are sequentially captured in the empty through holes). In the fine particle manipulating apparatus of the present invention, it is preferable to employ a power source that generates an AC voltage having no DC component. This is because, when an alternating voltage having a direct current component is applied, the fine particles move by receiving a force biased in a specific direction due to the electrostatic force generated by the direct current component, and are difficult to be captured in the through hole by the dielectrophoretic force. In addition, when an AC voltage having a DC component is applied, ions contained in the suspension containing fine particles cause an electrical reaction on the electrode surface and generate heat, which causes thermal movement of the fine particles, thereby controlling movement by dielectrophoretic force. This is because it becomes impossible to move to the through hole and capture it.

In the fine particle manipulating device of the present invention, as described above, it is preferable that only one fine particle can be captured in one through-hole by making the waveform of the applied AC voltage preferably rectangular. In order to achieve such an object, the arrangement, size, and shape of the through-holes and the size and shape of the through-holes are suitable for capturing only one fine particle in one through-hole. Is preferred. For example, in the present invention, when the through holes are preferably arranged in an array, it is difficult to achieve the above object even if the interval between adjacent through holes is too narrow or too wide. When the interval between adjacent through holes is narrow, the probability that a plurality of fine particles will be trapped in one through hole is high. Conversely, when the interval between adjacent through holes is wide, the interval between the through holes and the through holes is high. This is because there is a high probability that fine particles are left between them and a through hole that cannot capture the fine particles is generated. In order to avoid these, it is preferable that the interval between adjacent through holes is in the range of 0.5 to 6 times the particle size of the fine particles to be captured, and more preferably 1 to less than 2 times. It is particularly preferable that Furthermore, in order to be able to capture only one fine particle in one through hole, the diameter of the maximum circle inscribed in the opening on the accommodation portion side of the through hole is made smaller than the particle diameter of the fine particle to be captured. Alternatively, it is preferable that the diameter is in the range of 1 to 2 times the particle size of the fine particles and the depth of the through hole is less than 2 times the particle size of the fine particles. This is because if the inner diameter of the through-hole opening is larger than the particle size of the fine particles, the fine particles cannot close the through-holes, and the electric lines of force concentrate, and the other fine particles are attracted by the dielectrophoretic force. This is because the probability that two or more fine particles are trapped in the through hole is increased. Therefore, in order to capture only one fine particle in one through hole, the diameter of the maximum circle inscribed in the opening on the housing portion side of the through hole should be smaller than the particle diameter of the fine particle to be captured in the through hole. Is most preferred. In addition to this, even if the particle diameter is in the range of 1 to 2 times the particle diameter of the fine particles trapped in the through-hole and the depth of the through-hole is less than twice the particle diameter of the fine particle, one fine particle will be When trapped by, other fine particles are not drawn into the through hole even if they are attracted, so that the probability of trapping can be lowered. In addition, it is preferable that the opening shape (planar shape) of the through hole accommodating portion side is a shape having one or more corners in order to capture only one fine particle in one through hole. It is particularly preferable that the opening shape is a quadrilateral. Here, the corner is a portion where two sides constituting the shape of the through-hole intersect at an acute angle or an obtuse angle, and includes a shape in which the tip of the corner is slightly rounded. FIG. 16 illustrates a typical shape in which the opening shape of the through hole has at least one corner. Here, the quadrilateral means that the shape of the through hole has four corners (including a shape whose tip is slightly rounded). The four sides may be straight lines, or any of the four sides or any of the four sides may be slightly curved toward the center or outside of the through hole. FIG. 17 illustrates a typical shape of a quadrilateral through hole. In this way, if the opening shape of the through hole has a corner in part, the electric force lines are concentrated in the corner portion, the dielectrophoretic force is increased, and the fine particles are moved (pulled) more strongly. As a result, the probability that the fine particles are trapped in the through holes is improved. Although it is sufficient that at least one corner exists in the through hole, it is more preferable that a plurality of corners exist. However, an acute angle is easier to concentrate electric lines of force than an obtuse angle, and the dielectrophoretic force can be increased. Therefore, a polygon with a square or less is more preferable than a polygon with a pentagon or more. Further, even if it is a quadrangle, the specific shape is not particularly limited, and various shapes such as a trapezoid, a rhombus, and a parallelogram as shown in FIG. 17 can be selected. Among them, the shape of the quadrilateral through hole is almost equal in length of the four sides connecting the four corners, and the shape that is point symmetric at an angle of 90 degrees at the center of the through hole is the four sides of the quadrilateral through hole. The dielectrophoretic forces generated in the four corners are equal, and the dielectrophoretic forces generated in the four corners are also equal, and the distribution of the dielectrophoretic forces is point-symmetric so that the dielectrophoretic force with less bias is applied regardless of the position of the fine particles with respect to the through holes. Is particularly preferable. As such a shape, a square or a shape close to a square as shown in FIGS. 17A to 17D can be exemplified.
Next, a fine particle manipulation method using the fine particle manipulation device of the present invention will be described with reference to FIGS. As shown in FIG. 18, on the upper surface (surface on the side of the space 34 formed in the spacer 16 forming the accommodating portion) of the electrode substrate 15 forming the fine particle manipulating device, a pair of electrodes composed of an electrode a29 and an electrode b30 Are arranged in a comb shape, and when an AC voltage is applied between the electrode a and the electrode b by the AC power source 4, the electric lines of force 12 are concentrated in the through-hole 9 that is located directly above the electrode and penetrates in the vertical direction. Dielectrophoretic force acts on the fine particles 18 (see Patent Document 2). Here, the dielectrophoretic force includes a dielectrophoretic force (hereinafter referred to as a positive dielectrophoretic force 37) that acts to move the fine particles 18 to the through-holes 9 as shown in FIG. As shown in FIG. 4, there is a dielectrophoretic force (hereinafter referred to as a negative dielectrophoretic force 38) that moves the fine particles 18 so as to be detached from the through hole 9. When the positive dielectrophoretic force 37 acts, the fine particles 18 are trapped in the through-hole 9 as shown in FIG. 20, and when the negative dielectrophoretic force 38 acts, the fine particles 18 as shown in FIG. It is captured by the portion between the through hole and the through hole of the housing portion (on the insulator 8 in the apparatus shown in FIG. 5).

  The positive dielectrophoretic force and the negative dielectrophoretic force can be controlled by the dielectric constant of the target fine particles, the dielectric constant of the fine particle suspension 17 containing the fine particles, and the frequency of the alternating voltage used. For example, when the microparticles are mouse myeloma cells having a particle size of about 10 μm and the suspension is a 300 mM mannitol aqueous solution, the positive dielectrophoretic force acts on the mouse myeloma cells if the AC voltage frequency is 50 kHz or more. If it is less than 10 kHz, a negative dielectrophoretic force acts on mouse myeloma cells. Further, for example, when the fine particles are polystyrene fine particles having a particle diameter of about 6 μm and the suspension is pure water, the negative dielectrophoretic force acts on the polystyrene fine particles when the frequency of the AC voltage is 1 MHz or more and is positive at less than 10 Hz. The dielectrophoretic force acts on the polystyrene microparticles.

  The reason why the positive dielectrophoretic force and the negative dielectrophoretic force are switched at a certain frequency will be described. The dielectrophoretic force in the through-hole of the present invention is generally expressed by the following formula 1 depending on the particle radius, the AC voltage, the distance between the electrodes, the dielectric constant of the fine particle, and the dielectric constant of the suspension.

ここで、微粒子の誘電率と懸濁液の誘電率は、一般に交流電圧の周波数に依存して変化する。従って、交流電圧の周波数を変化させたときの微粒子の誘電率と懸濁液の誘電率の大小により、誘電泳動力の符号が決まる（変化する）。すなわち、交流電圧の周波数を変化させたとき、微粒子の誘電率が懸濁液の誘電率よりも大きくなれば、微粒子には正の誘電泳動力が作用し、微粒子の誘電率が懸濁液の誘電率よりも小さくなれば微粒子には負の誘電泳動力が作用する。このように、理論的には計算によって微粒子の誘電率と懸濁液の誘電率が等しくなる周波数（正の誘電泳動力と負の誘電泳動力が変化する境界の周波数、以下、境界周波数という）を求めることができる。しかし、微粒子の誘電率や懸濁液の誘電率を周波数ごとに測定することは煩雑であるため、微粒子の移動する方向の変化を観察することで、境界周波数を実験的に決定することが簡便である。このようにして決定される境界周波数は、当然、微粒子の種類や懸濁液の種類によって異なるものとなる。本発明の微粒子操作装置では、操作しようとする微粒子が貫通孔に捕捉されるように移動させるか、又は、微粒子が貫通孔から脱離するように移動させるように誘電泳動力が作用する方向を変化させるべく、適宜、電極に付加する交流の周波数を変えることが好ましい。この意味において、本発明の微粒子操作装置に採用する交流電源は、その周波数を任意に変更し得るものが好ましい。なお本発明の微粒子操作装置において、電極ａと電極ｂの間に、貫通孔から微粒子を脱離させる方向の負の誘電泳動力が作用するような周波数の交流電圧を印加すると、貫通孔ではなく、貫通孔と貫通孔の間、即ち収容部内壁を構成する部分に微粒子を捕捉することが可能となる。

Here, the dielectric constant of the fine particles and the dielectric constant of the suspension generally vary depending on the frequency of the AC voltage. Therefore, the sign of the dielectrophoretic force is determined (changed) depending on the dielectric constant of the fine particles and the dielectric constant of the suspension when the frequency of the AC voltage is changed. That is, when the frequency of the AC voltage is changed, if the dielectric constant of the fine particles becomes larger than the dielectric constant of the suspension, a positive dielectrophoretic force acts on the fine particles, and the dielectric constant of the fine particles becomes smaller than that of the suspension. If it becomes smaller than the dielectric constant, a negative dielectrophoretic force acts on the fine particles. Thus, theoretically, the frequency at which the dielectric constant of the fine particles and the dielectric constant of the suspension become equal by calculation (the boundary frequency where the positive and negative dielectrophoretic forces change, hereinafter referred to as the boundary frequency) Can be requested. However, since it is complicated to measure the dielectric constant of the fine particles and the dielectric constant of the suspension for each frequency, it is easy to experimentally determine the boundary frequency by observing changes in the moving direction of the fine particles. It is. The boundary frequency determined in this way naturally varies depending on the type of fine particles and the type of suspension. In the fine particle manipulating apparatus of the present invention, the direction in which the dielectrophoretic force acts so that the fine particle to be manipulated is moved so as to be captured by the through hole or the fine particle is moved so as to be detached from the through hole. In order to change, it is preferable to change the frequency of the alternating current applied to the electrode as appropriate. In this sense, the AC power source employed in the fine particle manipulation device of the present invention is preferably one that can arbitrarily change its frequency. In the fine particle manipulator of the present invention, when an AC voltage having a frequency at which a negative dielectrophoretic force in the direction of detaching the fine particles from the through hole is applied between the electrode a and the electrode b, not the through hole. The fine particles can be captured between the through holes, that is, in the portion constituting the inner wall of the accommodating portion.

  By using the fine particle manipulating apparatus of the present invention, as described above, by selecting the AC frequency as appropriate, only fine particles having a predetermined dielectric constant are selected from the suspension containing various fine particles, and the through holes are formed. It becomes possible to capture. Moreover, since the fine particle manipulation device of the present invention preferably has an electrode substrate installed in the lower part of the housing portion, a micropipette or the like is inserted from the through hole in a state where the fine particles are captured in the through hole by applying an AC voltage. It is possible to collect the fine particles captured by using. FIG. 23 is a conceptual diagram when a micropipette is employed as the fine particle manipulating device of the present invention and the fine particle collecting means 21 for collecting the fine particles 18 captured in the through holes 9. When selecting and collecting the target fine particles, the microscope 22 is installed as shown in FIG. 23, and the micropipette 21 is operated under the observation with the microscope to collect the fine particles. By adopting a microscope stage 40 that can be precisely positioned around 1 μm, the micropipette 21 can reliably collect fine particles to be collected while observing with a microscope. Further, when collecting the fine particles, if an AC voltage having a frequency at which a positive dielectrophoretic force that traps the fine particles in the through-hole acts between the electrodes a and b is applied from the power source 4, the target fine particles are obtained. It is possible to collect only the micropipette and leave other fine particles captured in the through-holes.

  FIG. 24 shows an application example of the particulate manipulation device of the present invention. For example, a suspension in which the presence of abnormal cells 41 such as cancer is suspected is applied to a fine particle manipulation device, and the cells are captured in the through holes. On the other hand, the marker molecule 42 that specifically binds to the surface of the abnormal cell 41 to be detected is modified with a fluorescent dye 43 or the like, and the marker molecule is introduced into the accommodating portion to specifically identify the marker molecule and the abnormal cell. The binding reaction. After discharging the suspension in the container together with unreacted marker molecules while capturing the cells in the through-holes, whether or not the marker molecules have been introduced into the cells captured in the through-holes, or Then, after being collected by the fine particle collecting means, it is detected by using a microscope or the like, or by using a known reagent and an optical device for detecting a fluorescent dye or the like. This makes it possible to collect or detect abnormal cells such as cancer by the fine particle manipulation device of the present invention.

  In the above description, the micropipette has been described as the fine particle collecting means. However, the fine particle collecting means is not particularly limited as long as the fine particles can be collected. In addition to the micropipette, fine particles that can accurately collect fine particles using an electroosmotic flow. Collection means can be used.

The fine particle manipulation device of the present invention has the following effects.
(1) The fine particle manipulating device of the present invention can be provided at low cost because the device configuration can be simplified because all of the pair of electrodes are installed on one electrode substrate, and because it is simple. In addition, since the configuration is simple, there is no need for cost and skill for maintenance and management.
(2) The fine particle manipulating apparatus of the present invention can quickly capture approximately one fine particle in one through hole. In particular, in an aspect in which a part of the housing portion is configured by an insulator having a plurality of through holes formed in an array, a plurality of fine particles can be quickly captured in the through holes arranged in an array.
(3) The fine particle manipulating apparatus of the present invention can improve the probability that approximately one fine particle can be captured in one through hole. In particular, in an aspect in which a part of the housing portion is formed of an insulator having a plurality of through holes formed in an array shape, the probability of capturing a plurality of fine particles in the through holes arranged in an array one by one can be improved. it can.
(4) The fine particle manipulating apparatus of the present invention can quickly detach (take out) the fine particles trapped in the through hole from the through hole.
(5) The fine particle manipulating apparatus of the present invention can easily desorb and collect arbitrary fine particles from the through hole while capturing the fine particles in the through hole.
(6) Since the fine particle manipulating apparatus of the present invention can shorten the distance between the electrodes, an electric field capable of sufficiently manipulating the fine particles can be obtained even with a relatively small and inexpensive AC power source.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to an Example.

  In Example 1, the apparatus main body 13 includes an accommodating portion (configured by the spacer 16 and the insulator 8 having the through holes 9 formed therein) and the electrode substrate 15, and the particulate manipulation apparatus shown in FIG. It was used.

  As the electrode substrate 15, a glass substrate having a length of 70 mm × width of 40 mm × thickness of 1 mm was used. The spacer 16 was used by hollowing out the center (34) of a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1.5 mm into a length of 20 mm and a width of 20 mm. Moreover, as shown in FIG. 8, the inlet 19 and the outlet 20 for introducing and discharging the suspension containing the fine particles were provided. The insulator 8 having the plurality of through holes 9 and the comb-like electrode 31 were integrally formed on the electrode substrate 15 by the photolithography and etching methods shown in FIGS.

  As shown in FIG. 25, Cr having a thickness of 1 nm was formed on one surface of glass 24 by sputtering (44), and Au having a thickness of 150 nm was further formed thereon by sputtering (45). Note that Cr is deposited to increase the adhesion between Au and glass. Next, a resist (25) is applied on the formed Au using a spin coater so as to have a film thickness of 1 μm, naturally dried for 1 minute, and then pre-baked (105 ° C., 15 minutes) using a hot plate. went. A positive resist was used.

  Next, using an exposure photomask 26 in which a comb-like electrode pattern is formed in which an electrode a having a width of 10 μm and an electrode b having a width of 10 μm are formed at an interval of 50 μm in an area of 30 mm in length × 30 mm in width, using a UV exposure machine The resist was exposed to light 27 and developed with developer 33. The exposure time and development time were adjusted so that the film thickness peeled off by development was 1 μm, which was equal to the resist film thickness, so that Au was exposed on the bottom surface of the through hole. After the development, the exposed Au film was stripped with 3% ammonium iodide iodide solution 49, and then the Cr film exposed after stripping the Au film with 30% diammonium cerium nitrate solution 50 was stripped. Finally, the resist was peeled off with a remover to form a comb-like electrode 31.

  On the comb-shaped electrode 31 on the electrode substrate thus fabricated, a resist 25 is applied using a spin coater so as to have a film thickness of 5 μm as shown in FIG. Was pre-baked (65 ° C., 1 minute → 95 ° C., 3 minutes). An epoxy negative resist was used as the resist. Next, a circular through-hole pattern with a diameter of 8.5 μm arranged in an array of 1000 vertical × 1000 horizontal in an area of 30 mm long × 30 mm wide with a vertical and horizontal interval between the through holes of 30 μm. The resist was exposed to light 27 with a UV exposure machine using the photomask 46 for exposure, and developed with the developer 33. The exposure time and the development time were adjusted so that the depth of the through hole was 5 μm, which was equal to the film thickness of the resist, so that the comb-like electrode on the bottom surface of the through hole was exposed. After development, post-baking (150 ° C., 15 minutes) was performed using a hot plate, the resist was hardened, and a lower substrate on which a comb-like electrode integrated with an insulator having a through hole was formed was manufactured.

  The electrode substrate 15 and the spacer 16 produced in this way were laminated and pressure bonded as shown in FIG. FIG. 9 is a B-B ′ sectional view of the cell fusion container shown in FIG. 8. The surface of the silicon sheet was sticky, and each component was brought into close contact by pressure bonding, and the suspension containing fine particles could be put into the main body 13 without leakage. Since the area formed by hollowing out the spacer is 20 mm long × 20 mm wide, the number of through holes existing in the space 34 is about 400,000. Moreover, the power supply (signal generator) which applies a voltage between electrodes was connected with the lead wire.

As the microparticles, mouse myeloma cells (particle size: about 10 μm) were used, suspended in an aqueous mannitol solution having a concentration of 300 mM, and the cell suspension was adjusted to a density of 0.7 × 10 ⁶ cells / mL.

  In order to evaluate the hydrophilicity of the insulator 8 formed with a through hole constituting a part of the housing portion, pure water is dropped on the surface, and the droplets and the insulator formed on the surface of the insulator at that time As a result of measuring the contact angle with the surface, the contact angle was about 74 ° and was hydrophobic. Therefore, in order to hydrophilize the insulator 8 in which the through hole is formed, the electrode substrate 15 (the insulator 8 in which the through hole is formed is integrally formed on the electrode substrate 15) is replaced with BSA (1 mg / mL). The BSA was physically adsorbed on the surface of the insulator by immersing in the contained 300 mM mannitol aqueous solution for about 1 hour. Similarly, after BSA is physically adsorbed, pure water is dropped on the surface of the insulator, and the contact angle between the droplet formed on the surface of the insulator and the surface of the insulator is measured. Was about 37 °, confirming that it was made hydrophilic. In this case, the affinity between the insulator surface and the mouse myeloma cell (having hydrophilicity) to be manipulated is relatively high.

  600 μL of the cell suspension (number of cells: about 400,000) is injected from the introduction port of the spacer 16 using a syringe, and a rectangular wave AC voltage having a voltage of 2 Vpp and a frequency of 3 MHz is applied between the electrodes as an AC voltage by a signal generator. When applied, one cell could be captured in one through-hole formed in an array in an extremely short time of about 2 to 3 seconds, and the cells could be arranged in an array. Note that “captured” means both the case where fine particles enter the through-hole and the case where fine particles remain at the edge of the through-hole, and the same definition applies to the following examples and comparative examples. did. At this time, the capture rate of fine particles in which approximately one cell enters one through-hole was about 90%. The fine particle capture rate is the number of through-holes containing one fine particle when the fine particle is introduced and captured so that 225 through holes of 15 vertical x 15 horizontal are visible in the field of view of the microscope. It is defined by a value divided by the number of 225 through holes, and the same applies to the following examples and comparative examples.

  As shown in FIG. 23, the fine particle collecting means 21 is installed in the main body 13. As the fine particle collecting means, using a pipette capable of accurately collecting fine particles using electroosmotic flow, it was possible to collect specific cells captured in the through-hole while observing with the microscope 22. Further, by continuously applying an alternating voltage (2 Vpp, 3 MHz) between the comb electrodes by the power source 4 and causing the dielectrophoretic force to continue to act in the direction of capturing the cells in the through-holes, cells other than the collected cells can pass through the through-holes. However, no desorption was confirmed.

  Subsequently, when a sine wave AC voltage having a voltage of 2 Vpp and a frequency of 1 kHz was applied between the electrodes as an AC voltage by a signal generator, the cells trapped in the arrayed through holes were penetrated in an extremely short time of about 2 to 3 seconds. It was easily removed from the hole. When the cell suspension was taken out by tilting the main body, the number of taken out cells was about 80% of the number of introduced cells.

(Comparative example)
For comparison, operation was performed using the fine particle manipulating apparatus shown in FIG. In the fine particle manipulation device of FIG. 10, the main body 13 has a configuration in which a housing portion (consisting of an insulator 8 having a spacer 16 and a through hole) is sandwiched between an upper electrode substrate 47 and a lower electrode substrate 48. . As the upper electrode substrate and the lower electrode substrate, a glass substrate having a length of 70 mm, a width of 40 mm, and a thickness of 1 mm was formed by depositing ITO (film thickness: 150 nm). The spacer 16 was used by hollowing out the center (34) of a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1.5 mm into a length of 20 mm and a width of 20 mm. Further, as shown in FIG. 10, an introduction port 19 and a discharge port 20 for introducing and discharging a suspension containing fine particles were provided. The insulator 8 having the plurality of through holes 9 and the electrode were integrally formed on the electrode substrate by the photolithography and etching methods shown in FIGS.

  First, ITO (23) is formed on one surface of the glass 24, and a resist (25) is applied to the film forming surface so as to have a film thickness of 5 μm using a spin coater. After naturally drying for 1 minute, a hot plate is attached. And prebaked (65 ° C., 1 minute → 95 ° C., 3 minutes). An epoxy negative resist was used as the resist.

  Next, a circular through-hole pattern with a diameter of 8.5 μm arranged in an array of 1000 vertical × 1000 horizontal in an area of 30 mm long × 30 mm wide with a vertical and horizontal interval between the through holes of 30 μm. The resist was exposed to light 27 with a UV exposure machine using the photomask 46 for exposure, and developed with the developer 33. The exposure time and development time were adjusted so that the depth of the through hole was 5 μm, which was equal to the resist film thickness, so that ITO was exposed on the bottom surface of the through hole. After the development, post baking (150 ° C., 15 minutes) was performed using a hot plate, the resist was hardened, and the lower electrode substrate 48 integrally formed with the insulator 8 having the through holes was manufactured.

  The lower electrode substrate 48 and the spacer 16 produced in this way were laminated and pressure bonded as shown in FIG. Further, the pressure-bonded product and the upper electrode substrate 47 were detachably pressed with a metal fitting. FIG. 11 is a C-C ′ cross-sectional view of the cell fusion container shown in FIG. 10. The surface of the silicon sheet was sticky, and each component was brought into close contact by pressure bonding, and the suspension containing fine particles could be put into the main body 13 without leakage. Since the area formed by hollowing out the spacer is 20 mm long × 20 mm wide, the number of through holes existing in the space 34 is about 400,000. Moreover, the power supply (signal generator) which applies a voltage between electrodes was connected with the lead wire.

As the microparticles, mouse myeloma cells (particle size: about 10 μm) were used, suspended in an aqueous mannitol solution having a concentration of 300 mM, and the cell suspension was adjusted to a density of 0.7 × 10 ⁶ cells / mL.

  In order to evaluate the hydrophilicity of the insulator 8 formed with a through hole constituting a part of the housing portion, pure water is dropped on the surface, and the droplets and the insulator formed on the surface of the insulator at that time As a result of measuring the contact angle with the surface, the contact angle was about 74 ° and was hydrophobic. Therefore, in order to hydrophilize the insulator 8 in which the through hole is formed, the lower electrode substrate 48 (the insulator 8 in which the through hole is formed is integrally formed on the lower electrode substrate 48) is replaced with BSA ( The BSA was physically adsorbed on the surface of the insulator by immersing in a 300 mM mannitol aqueous solution containing 1 mg / mL for about 1 hour. Similarly, after BSA is physically adsorbed, pure water is dropped on the surface of the insulator, and the contact angle between the droplet formed on the surface of the insulator and the surface of the insulator is measured. Was about 37 °, confirming that it was hydrophilic. In this case, the affinity between the insulator surface and the mouse myeloma cell (having hydrophilicity) to be manipulated is relatively high.

  600 μL of the cell suspension (number of cells: about 400,000) is injected from the introduction port of the spacer 16 using a syringe, and a rectangular wave AC voltage having a voltage of 2 Vpp and a frequency of 3 MHz is applied between the electrodes as an AC voltage by a signal generator. Although applied, cells could not be trapped in the through hole. This is because the distance between the upper electrode substrate and the lower electrode substrate is as long as 1.5 mm, and a voltage of 2 Vpp can provide a sufficient electric field for generating a dielectrophoretic force for trapping cells in the through-hole. It was because it was not possible.

  Next, when a rectangular wave AC voltage having a voltage of 10 Vpp and a frequency of 3 MHz is applied as an AC voltage by a signal generator, one cell can be captured in one through-hole formed in an array in about 2 to 3 seconds. did it. However, since there is an upper substrate in the main body 13 used in this comparative example, when collecting any cell trapped in the through-hole, the application of voltage is stopped and the upper electrode substrate is removed by removing the metal fittings. There was an example in which a cell that had been trapped once detached from the through-hole had to be removed.

  Using the same fine particle manipulation apparatus as in Example 1, mouse myeloma cells similar to those in Example 1 were introduced into the fine particle manipulation region, and the frequency and the rectangular wave AC voltage application time were kept as described above, and only the frequency was reduced. As a result, as shown in FIG. 31, the cell capture rate was about 81% at 50 kHz, and about 50% of cells could be captured at 30 kHz. However, almost no cells could be captured at 10 kHz. From this result, it can be seen that the AC frequency of the AC power source for capturing cells in the through-hole is 30 kHz or more, preferably 50 kHz or more.

  In addition, the cell detachment rate was measured using the same method. The cell detachment rate is a value obtained by dividing the number of cells extracted from the through-hole among mouse myeloma cells captured using the first AC power supply after introducing the suspension, and divided by the number of captured cells. Defined. When a rectangular wave AC voltage having a voltage of 2 Vpp and a frequency of 10 kHz was applied between the electrodes by an AC power source for 10 seconds, cells captured in the plurality of through holes formed in an array could be taken out. At this time, the detachment rate of the cells was about 100%. When the voltage and the rectangular wave AC voltage application time were kept as described above, only the frequency was increased. As shown in FIG. 32, the desorption rate was about 100% at 10 kHz, and about 50% was desorbed at 20 kHz. However, it hardly desorbed at 100 kHz. From this result, it can be seen that the AC frequency of the AC power source for detaching cells from the through-hole is desirably less than 20 kHz, preferably less than 10 kHz.

Using the same fine particle manipulating apparatus as in Example 1, commercially available polystyrene fine particles (particle size: about 6 μm, polystyrene fine particle concentration: 2.5%) were used as fine particles, suspended in pure water and 0.8 × 10 ⁶ particles / The polystyrene fine particle suspension was adjusted to a density of mL. However, unlike Example 1, the hydrophilic treatment of the insulator 8 in which the through holes were formed was not performed. That is, since the insulator 8 is hydrophobic, the affinity between the polystyrene fine particles exhibiting hydrophobicity and the insulator is relatively high.

  500 μL of the above-mentioned polystyrene fine particle suspension (number of fine particles: about 400,000) is injected from the introduction port of the spacer using a syringe, and a rectangular wave AC voltage with a voltage of 2 Vpp and a frequency of 9 Hz is applied between the electrodes as an AC voltage by a signal generator. When applied, approximately one polystyrene microparticle could be captured in one through-hole formed in an array in an extremely short time of about 2 to 3 seconds, and the microparticle capture rate was about 60%.

  Next, when a sine wave AC voltage having a voltage of 2 Vpp and a frequency of 100 kHz was applied between the electrodes as an AC voltage by a signal generator, the polystyrene fine particles captured in the arrayed through-holes in an extremely short time of about 2 to 3 seconds. Could be removed from the through hole. Subsequently, when the main body 13 was tilted and the polystyrene fine particle suspension was taken out, the number of polystyrene fine particles taken out was about 78% of the number of introduced polystyrene fine particles.

  As a result of further examination of the frequency of the alternating voltage and the capture and removal of polystyrene fine particles in the through holes using the fine particle manipulation measures used in Example 3, the frequency of the alternating voltage (voltage 2 Vpp) is less than about 100 Hz. The polystyrene fine particles started to be captured in the through holes, and when the frequency became less than 10 Hz, about 50 to 60% of the polystyrene fine particles were captured in the through holes. Further, when the frequency of the AC voltage is about 1 kHz or more, polystyrene fine particles begin to be desorbed from the through hole. When the frequency becomes 1 MHz or higher, approximately 90% or more of the polystyrene fine particles trapped in the through hole are desorbed from the through hole. I was able to.

It is a figure for demonstrating the invention described in patent document 2. FIG. It is a figure for demonstrating the invention described in patent document 2. FIG. It is a figure for demonstrating the invention described in patent document 2. FIG. It is a figure for demonstrating the invention described in patent document 2. FIG. It is a figure for demonstrating the fine particle operating device of this invention. It is AA 'sectional drawing of the main body 13 of the microparticle operation apparatus shown in FIG. It is the figure which looked at arrangement | positioning of a pair of electrode and through-hole on the electrode substrate which comprises the apparatus shown in FIG. 5 from the upper side. It is a figure for demonstrating the fine particle operating device of this invention. It is BB 'sectional drawing of the main body 13 of the microparticle operation apparatus shown in FIG. It is a figure for demonstrating the fine particle operation apparatus used as a comparative example. It is CC 'sectional drawing of the main body 13 of the microparticle operation apparatus shown in FIG. It is a figure which shows a sine wave among the waveforms of the alternating voltage used for this invention. It is a figure which shows a triangular wave among the waveforms of the alternating voltage used for this invention. It is a figure which shows a trapezoid wave among the waveforms of the alternating voltage used for this invention. It is a figure which shows a rectangular wave among the waveforms of the alternating voltage used for this invention. It is a figure which shows the example of the through-hole shape in this invention. It is a figure which shows the example of the through-hole shape in this invention. It is a figure for demonstrating operation by the fine particle operating device of this invention. It is a figure for demonstrating operation by the fine particle operating device of this invention. It is a figure for demonstrating operation by the fine particle operating device of this invention. It is a figure for demonstrating operation by the fine particle operating device of this invention. It is a figure for demonstrating operation by the fine particle operating device of this invention. It is the figure of the apparatus which installed the micropipette as a microparticles | fine-particles operation apparatus of this invention, and the microparticles | fine-particles collection means which extract | collects the specific microparticles | fine-particles caught by the through-hole. It is a figure which shows the example which applied the fine particle operation apparatus of this invention as a detection apparatus of an abnormal cell. It is the schematic of the process of producing a comb-shaped electrode using general photolithography and etching. It is the schematic of the process of producing a through-hole using common photolithography and etching.

1: Fusion area 2: Electrode 3: Conductive wire 4: Power supply 5: AC power supply 6: DC pulse power supply 7: Switch 8: Insulator 9: Through hole 10: Cell A
11: Cell B
12: Electric field line 13: Main body 14: Upper lid 15: Electrode substrate 16: Spacer 17: Fine particle suspension 18: Fine particle 19: Inlet 20: Discharge port 21: Fine particle operating means 22: Microscope 23: ITO
24: Glass 25: Resist 26: Photomask for exposure depicting a comb-like electrode pattern 27: Exposure 28: Lower electrode formed integrally with an insulator having a through hole 29: Electrode a
30: Electrode b
31: Comb electrode 32: Center axis of through-hole 33: Developer 34: Space formed in spacer 16 35: Center axis of conductive member 36: Peak voltage 37: Positive dielectrophoretic force 38: Negative dielectrophoretic force 39: Fine particle manipulation device 40: Stage 41: Abnormal cell 42: Marker molecule 43: Fluorescent dye 44: Cr
45: Au
46: Photomask for exposure depicting a through-hole pattern 47: Upper electrode substrate 48: Lower electrode substrate 49: 3% ammonium iodide iodide solution 50: 30% diammonium nitrate cerium solution 51: Fusion cell

Claims (9)

A housing part for housing the fine particle suspension; an electrode substrate on which a pair of electrodes are arranged; and an AC power source connected to the electrodes. A part of the housing part is made of an insulating material and has the suspension. A fine particle manipulating apparatus having a through-hole that allows a suspended liquid to contact each electrode. The pair of electrodes is composed of two parallel electrodes extending from each of the two opposing sides toward the other side, or extends from each of the two opposing sides toward the other side. The fine particle manipulating apparatus according to claim 1, comprising three or more parallel and equally spaced electrodes. The electrode substrate is disposed at a lower portion of the housing portion, and the lower portion of the housing portion is made of an insulating material and has a vertical through hole that allows the suspension to contact the electrodes. The fine particle manipulating apparatus according to claim 1, characterized in that it is characterized in that: The said accommodating part is comprised by the insulator which has the through-hole of a perpendicular direction arrange | positioned under the frame which hold | maintains the said suspension, and the said frame, From the Claim 1 characterized by the above-mentioned. 4. The fine particle manipulation device according to any one of items 3. The fine particle manipulating apparatus according to any one of claims 1 to 4, wherein the hole of the accommodating portion has a shape and a dimension capable of capturing one fine particle. 6. The fine particle manipulating apparatus according to claim 5, wherein a diameter of a maximum circle inscribed in the opening of the hole is smaller than a particle diameter of the fine particles. The diameter of the maximum circle inscribed in the opening of the hole is in the range of 1 to 2 times the particle size of the fine particles, and the depth is less than 2 times the particle size of the fine particles. Item 6. The fine particle manipulation device according to Item 5. The fine particle manipulation device according to any one of claims 1 to 7, wherein the through holes are arranged in an array. 9. The fine particle manipulating apparatus according to claim 1, wherein a material of the insulator constituting a part of the housing portion is a material having affinity for the fine particles.

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