JP5047034B2 - Particle separation method and separation apparatus - Google Patents

Description

  The present invention relates to a method and apparatus for dielectrophoresis of a plurality of types of particles in a liquid and separating them using a difference in dielectric properties.

  Conventionally, as a method for separating particles, particularly cells and microorganisms, present in a liquid, there is a method in which the liquid is caused to flow through a flow path in which electrodes are arranged and separated using a fluid action and dielectrophoresis. In dielectrophoresis, when a particle is placed in a non-uniform alternating electric field, positive and negative polarization occurs in the particle, and if the dielectric constant of the medium surrounding the particle is greater than that of the material, the material moves in the direction of lower electric field. When the dielectric constant of the medium is smaller than that of the particles, the particles move in a direction in which the electric field is strong. This separation method using dielectrophoresis is used, for example, in the medical industry to remove bacteria, microorganisms, viruses, and the like from pharmaceuticals and body fluids, or to remove contaminants such as dialysis.

  For example, in Patent Document 1, the difference in the dielectrophoretic force of particles flowing at a constant speed is used to separate particles from the time difference to reach the outlet.

Patent Document 2 discloses a method of increasing the separation accuracy by changing the dielectric characteristics of specific particles using an antibody when the particles contained in the liquid are known. Dielectric properties are selectively changed by capturing a plurality of types of particles present in a solution once on an electrode and binding antibodies to specific particles. Thereafter, by applying a flow action, only the particles specifically bound to the antibody are flowed to improve the separation accuracy.
JP 7-505717 A (Patent No. 3182151) JP 2003-223 A (Patent No. 3896686)

  However, in the method described in Patent Document 1, the flow velocity near the wall surface on which the electrodes are arranged is slow, so that the flow effect is weak against particles existing near the wall surface. Furthermore, when the material of the particles to be separated is similar (for example, cells and microorganisms), the difference in the dielectrophoretic force is considered to be small because there is no significant difference in dielectric properties. Therefore, depending on the position in the flow path, that is, when the particles flow near the wall where the electrodes exist, the effect of the flow on the particles becomes weak. Is difficult and the separation accuracy is reduced.

  Further, in the method described in Patent Document 2, when the difference in the dielectrophoretic force of particles contained in the liquid is small and the type is unknown, it is difficult to select an appropriate antibody, and the separation accuracy is improved. It is difficult to let In addition, since an antigen-antibody reaction is used, it takes time and effort for separation.

  In view of the above, there has been a demand for a method that can easily and accurately separate substances other than the target without trapping substances other than the target even when unknown particles are contained in the liquid. The present invention was invented in view of the above-mentioned problems in the prior art, and an object of the present invention is to provide a method for accurately separating a plurality of types of particles contained in a liquid. In particular, even when the difference in dielectrophoretic force is small, such as when the particles are similar, a method that improves the separation accuracy with a simple method, regardless of the type of particles contained in the liquid. The purpose is to provide.

Therefore, the method for separating particles according to the present invention is as follows.
A method in which at least two kinds of particles contained in a liquid flowing in a flow path are dielectrophoresed by a non-uniform electric field generated by an electrode and separated using a difference in dielectric properties,
Flowing the particles downstream from the uneven distribution of the particles by the uneven distribution electrode that generates the first non-uniform electric field;
A trapping target is disposed on the downstream side of the unevenly-distributed electrode, on the opposite side of the particle-distributed side across the flow path, and is configured to generate a second non-uniform electric field. Capturing the particles;
A method for separating particles characterized by comprising:

Moreover, the particle separation apparatus according to the present invention comprises:
An apparatus for dielectrophoretic migration of at least two types of particles contained in a liquid and separating them using a difference in dielectric properties,
The device
A flow path for flowing the liquid;
Means for generating a flow action in the flow path;
An unevenly distributed electrode that is installed in the flow path, generates a first non-uniform electric field, and unevenly distributes the particles;
A capturing electrode that is located downstream of the uneven distribution electrode and on the opposite side of the side where the particles are unevenly distributed across the flow path, generates a second non-uniform electric field, and captures the particles to be captured;
It is characterized by having.

  According to the present invention, even when the difference in dielectrophoretic force between particles contained in a liquid is small, the separation accuracy can be improved regardless of the type of particles.

  Dielectrophoresis is a phenomenon in which particles move in a non-uniform electric field due to the interaction between the electric conductivity and dielectric constant of the particles, the electric conductivity and dielectric constant of the medium, and the applied frequency. The force acting on the particles is called dielectrophoretic force. In addition, the dielectrophoretic force is classified into two types, that is, a positive dielectrophoretic force in which particles move toward a stronger electric field and a negative dielectrophoretic force that moves toward a weaker electric field. When the frequency is selected according to the type of particle, it is possible to capture by applying a positive dielectrophoretic force or to eliminate it by applying a negative dielectrophoretic force. Hereinafter, a case where a positive dielectrophoretic force acts on particles will be described as an example.

  The particles placed in the electric field induce a positive polarization charge + q on the downstream side of the electric field and a negative polarization charge -q on the upstream side. Pulls this part upstream of the electric field. If the molecule is neutral, the absolute values of + q and -q are equal, and if the electric field is constant regardless of the location, the forces acting on the two are balanced and the molecule does not move. However, when the electric field is not uniform, the force drawn to the strong electric field side becomes larger, and the molecule is driven to the strong electric field side. That is, particles that are dielectrically polarizable in a non-uniform electric field have an effective polarizability of such particles that is different from the polarizability of the surrounding medium, even if such particles have no net charge. In some cases, it receives a dielectrophoretic force. The movement is determined not by the charge of the particles as in the electrophoresis phenomenon but by the dielectric properties (for example, conductivity and dielectric constant).

  Note that the electric field is oscillating at any point, and in an alternating electric field where the pattern is fixed, when the electric field fluctuates periodically, the dielectrophoretic force acting on the particles is oscillating and simple. Is tropic. Particles move in the direction of strong electric fields (positive dielectrophoresis) when the polarizability is higher than the medium in which such particles are suspended.

  The particle separation method of the present invention separates target particles using a difference in dielectrophoretic force between particles. That is, since the dielectrophoretic force depends on the dielectric properties of the particles, the target particles can be separated and captured by the difference in the dielectric properties of the particles. Examples of the element that causes the difference in the dielectric properties of the particles include the size of the particles, the dielectric constant, the surface charge, or the surface film property. The separation can be performed based on the difference in at least one of these elements.

Here, the present invention
A method in which at least two kinds of particles contained in a liquid flowing in a flow path are dielectrophoresed by a non-uniform electric field generated by an electrode and separated using a difference in dielectric properties,
Flowing the particles downstream from the uneven distribution of the particles by the uneven distribution electrode that generates the first non-uniform electric field;
A trapping target is disposed on the downstream side of the unevenly-distributed electrode, on the opposite side of the particle-distributed side across the flow path, and is configured to generate a second non-uniform electric field. Capturing the particles;
It is the separation method of the particle | grains characterized by having.

  In the method for separating particles according to the present invention, first, the particles are once unevenly distributed by the unevenly-distributing electrode before reaching the region of the capturing electrode. The particles contained in the liquid are unevenly distributed and then flowed to the capturing electrode, so that only the particles to be captured are attracted and captured by the capturing electrode, and particles other than the particles to be captured (hereinafter abbreviated as other particles). ) Can flow downstream without being captured. Since the particles in the liquid naturally flow through the flow path in a dispersed state, even if there is a difference in dielectrophoretic force between the particles to be captured and the other particles, other particles When the gas flows in the vicinity of the capturing electrode, it is captured by the capturing electrode. In particular, when the difference in dielectrophoretic force between the particles is small, other particles are also captured significantly together with the particles to be captured. On the other hand, in the present invention, before reaching the area of the capturing electrode, all particles are once distributed unevenly in the flow channel portion on the opposite side of the capturing electrode by the unevenly distributed electrode, so that other particles are captured. It can be prevented from being trapped by the electrode. Therefore, the particles can be effectively separated even when the difference in the dielectrophoretic force between the particles is small.

(particle)
In the present invention, particles are cells, cell aggregates, intracellular organelles, biological substances such as bacteria, viruses or nucleic acids, inorganic substances such as inorganic substances, crystals or synthetic particles, or liquids such as fine water droplets in oil. It is used as a concept that widely includes gases such as bubbles and bubbles. Examples of the biological substance include nucleotide chains, chromosomes, peptide chains, proteins, immunoglobulins, serum proteins, antibodies, antigens, lipids, sugar chains or microorganisms. Examples of the inorganic substance include metals such as silica, alumina, gold, titanium, iron or nickel, polysaccharides such as agarose, cellulose or insoluble dextran, or polymer compounds such as polystyrene or styrene-butadiene copolymer. It is done.

  Examples of the liquid to be subjected to the separation method according to the present invention include biological samples such as body fluids such as serum, plasma, spinal fluid, synovial fluid, and lymph, or excrement such as urine and feces, and processed products thereof. Etc. In addition, as a treated product, for example, a sample obtained by appropriately diluting these biological samples with water or a buffer solution, or particles obtained from these biological samples are appropriately dissolved or suspended in water or a buffer solution to reconstitute. And the like obtained.

  Moreover, in this specification, the particle | grains used as the capture | acquisition object are not restricted to one type, Two or more types may be sufficient. The same applies to particles other than the particles to be captured (other particles).

(electrode)
There are at least two types of electrodes used in the present invention: an unevenly distributed electrode and a capturing electrode.

  The unevenly distributed electrode generates a non-uniform electric field, and causes the particles flowing in the flow path to be unevenly distributed in the unevenly distributed portion near the unevenly distributed electrode by a dielectrophoretic force, for example, by a positive dielectrophoretic force. Located upstream of the capture electrode in the flow path. Here, the unevenly distributed portion means the surface of the unevenly distributed electrode and the vicinity thereof. Further, uneven distribution of particles means that the particles are retained or attracted to the uneven distribution portion.

  The capturing electrode generates a non-uniform electric field and captures particles flowing through the flow path to the capturing section by dielectrophoretic force, and is positioned downstream of the unevenly distributed electrode section in the flow path. Here, the capturing part means the surface of the capturing electrode and the vicinity thereof. Moreover, capturing particles means retaining the particles in the capturing part.

  Moreover, each said electrode may have a counter electrode which opposes on both sides of a flow path, respectively. The distance between each electrode and its counter electrode usually depends on the width of the channel. That is, the unevenly-distributed electrode and its counter electrode (referred to as an unevenly-distributed electrode portion) are arranged so as to sandwich the flow path, and the capture electrode and its counter electrode (referred to as a capture electrode section) are also sandwiched between the flow paths. Be placed. And the said electrode part for uneven distribution and the said electrode part for capture | acquisition are arrange | positioned adjacent to a flow path. Note that each of the electrodes may be arranged in the flow path as long as it has the effect of the present invention. For example, the electrodes may be arranged on the upper surface and the lower surface of the flow path, or on the side surface. Also good.

  In addition, each electrode part is formed so that the positions of the unevenly distributed part and the capturing part are opposite to each other across the flow path. By disposing each electrode portion in this manner, as described above, even when it is desired to separate particles having a small difference in dielectrophoretic force, particles to be captured can be captured effectively. In other words, even when the difference in the dielectrophoretic force between the particles is small, the particles in the flow path are once unevenly distributed in the unevenly distributed part by the unevenly distributed electrode part and then flowed to the downstream capturing electrode part. It is possible to prevent particles with weak force from approaching the capturing part, and to capture only particles having a strong dielectrophoretic force (particles to be captured) in the capturing part, thereby improving the separation accuracy. The particles separated and recovered by the separation method according to the present invention can be used for other processes such as analysis.

  The unevenly distributed electrode portion and the capturing electrode portion generate a non-uniform electric field by an alternating voltage. For example, it is desirable that the unevenly-distributing electrode and the capturing electrode have different areas and different widths from the counter electrode. That is, when the width and height of the flow path are constant and the electrodes are provided on both sides of the flow path side wall, the horizontal width can be adjusted only by adjusting the vertical width of each electrode to the height of the flow path. The area ratio with the counter electrode can be selected. By disposing counter electrodes with different areas (especially width sizes), it is possible to generate a significantly non-uniform electric field when a voltage is applied, and the particles have a smaller area (the electrode on the side where the electric field is stronger). ) Positive dielectrophoresis towards. The area ratio between the unevenly-distributing electrode and the capturing electrode and the respective counter electrode can be appropriately selected in consideration of the migration speed of particles to be captured and other particles. For example, the area ratio is desirably a ratio of 1: 4 to 1: 5 so that a non-uniform electric field in which particles are likely to migrate is generated, but a size ratio other than the above may be used to adjust the migration speed and the like. Good.

  Further, when the unevenly-distributing electrode and the capturing electrode are combined into a set, a plurality of sets of electrodes can be arranged in the flow path. By configuring in this way, it is possible to further improve the separation accuracy.

  The distance between the opposing electrodes can be adjusted as appropriate according to the type of particles. If the distance between the opposing electrodes is too large for the particles, a non-uniform electric field with sufficient electric field strength cannot be formed, and if too small, the particles may be unevenly distributed or trapped. . Although not particularly limited, for example, when the particles are blood cells or microorganisms, it is preferably 1 μm or more and 500 μm or less, more preferably 10 μm or more and 300 μm or less, and particularly preferably 100 μm or more and 200 μm or less. preferable.

  The distance between the unevenly-distributing electrode part and the capturing electrode part can be appropriately adjusted in consideration of the flow rate of the liquid and the particles contained in the liquid, particularly the particles to be captured. If the distance is too long, the effect of uneven distribution may be reduced, and if the distance is too short, a non-uniform electric field is generated between the electrodes on the same plane, so that the unevenly distributed side In some cases, particles may be supplemented as they are. Although not particularly limited, for example, when the particle is a blood cell or a microorganism, it can be usually 200 μm or more and 2000 μm or less, and preferably 500 μm or more and 1000 μm or less.

  As a material of the electrode, any material that is usually used as an electrode can be used without particular limitation. Examples thereof include stainless steel, copper, and aluminum.

  The electrode can be provided in a flow path made of a material such as glass, plastic, quartz, or silicon using a known fine processing technique.

  The electrode may be coated with an organic thin film or the like.

  The frequency and voltage applied to the electrode can be appropriately selected so that the separation accuracy is optimal in consideration of particles to be captured, other particles, liquid medium, and the like. And the capturing electrode portion may be different. Usually, a preliminary examination is performed, and an AC voltage having a single frequency selected optimally is applied to the electrodes. In addition, when the capture target is a cell or a microorganism, the frequency is preferably 100 kHz to 6.5 MHz. The induced electrophoretic force received by the particles is affected by the pH, conductivity, voltage frequency, etc. of the liquid, and therefore it is desirable to select the separation conditions in consideration of these. If the dielectrophoretic force applied to the particles to be separated can be divided into positive and negative depending on the frequency, for example, if the particles to be captured are positive and the other particles are negative, the particles to be captured are delayed. It will pass through the flow path, and the separation accuracy can be further improved. However, if there is no difference in dielectrophoretic force between particles, such as blood cells and microorganisms, a positive dielectrophoretic force is applied to certain blood cells and a negative dielectrophoretic force is applied to other microorganisms. It cannot be applied, and the positive / negative distribution of the dielectrophoretic force cannot be generalized for each type of particle. Therefore, the present invention is particularly effective in such a case.

  Further, the unevenly-distributed electrode section can also flow the unevenly distributed particles downstream of the flow path by turning the power supply on and off at regular intervals. By turning off the power supply of the unevenly-distributed electrode part at regular intervals, particles to be captured that are attracted to the unevenly distributed part can be flowed to the downstream capture electrode part, and the separation accuracy can be improved. It should be noted that whether the power is turned on or off, and the time and the like can be appropriately selected so that the separation conditions are optimal.

(Flow path)
A liquid containing particles to be separated, for example, a liquid in which two or more kinds of particles are dissolved or suspended in a non-uniform electric field formed by using the unevenly distributed electrode part and the capturing electrode part is allowed to flow through the flow path. Then, the particles to be captured are captured and separated. At least the unevenly distributed electrode and the capturing electrode are provided in the flow path. The channel width is not particularly limited, but at least the place where each of the electrode portions is arranged as described above corresponds to the distance between the electrodes facing each other.

  The shape of the flow path may be any shape such as a linear shape or an annular shape as long as the liquid containing the particles can flow. As the material of the flow path, any material that is generally used for forming the flow path, such as glass, quartz, plastic, or silicon, may be used, and a non-conductive material is desirable. As a means for generating the fluid action, any commonly used drive device such as a syringe pump or an electroosmotic flow pump may be used.

  In addition, the flow path can be provided with a portion for injecting or collecting a liquid containing particles. The liquid recovery tank can be appropriately selected according to the amount of liquid to be introduced and the properties of the particles, and any liquid recovery tank may be used as long as it is generally used as a container.

  An apparatus capable of suitably implementing the above-described particle separation method according to the present invention will be described below.

The particle separation apparatus according to the present invention comprises:
An apparatus for dielectrophoretic migration of at least two types of particles contained in a liquid and separating them using a difference in dielectric properties,
The device
A flow path for flowing the liquid;
Means for generating a flow action in the flow path;
An unevenly distributed electrode that is installed in the flow path, generates a first non-uniform electric field, and unevenly distributes the particles;
A capturing electrode that is located downstream of the uneven distribution electrode and on the opposite side of the side where the particles are unevenly distributed across the flow path, generates a second non-uniform electric field, and captures the particles to be captured;
It is characterized by having.

  Using the particle separation apparatus according to the present invention, particles contained in a liquid can be effectively separated. In particular, even when there is no difference in dielectrophoretic force between particles, separation can be performed effectively.

  The combination of particles to be captured and other particles is not particularly limited, and examples thereof include animal cells and microorganisms, particularly blood cells and pathogenic bacteria. As conditions for the dielectrophoresis, the frequency is preferably 100 kHz to 6.5 MHz, the voltage is 10 Vpp or more, and the solvent conductivity is preferably 0 to 0.13 [S / m]. Depending on the conditions, conditions other than those described above may be used.

  Moreover, as described above, the particle separation apparatus according to the present invention can arrange a plurality of sets of the unevenly-distributing electrode and the capturing electrode in the flow path. As an example, FIG. 11 shows an apparatus configuration when the number of sets is four.

  The wiring 31 for the unevenly distributed electrode part becomes an electrode constituting each unevenly distributed electrode part, and the wiring 32 for the capturing electrode part becomes an electrode constituting each capturing electrode part. In the example shown in FIG. 11, the unevenly distributed electrode portions are managed by the same power source. However, the present invention is not limited to this, and each unevenly-distributed electrode portion can be managed separately. However, in terms of the configuration of the apparatus, it is preferable that each unevenly distributed electrode portion is collectively managed by one power source. The same applies to the capturing electrode section.

  The width and height of the flow path 33 can be appropriately adjusted according to the particles and liquid to be separated. In addition, whether the electrodes are arranged on the upper and lower wall surfaces or the side walls varies depending on the method of installing the device and how to grasp the device configuration, and is not particularly limited. Reference numeral 34 denotes the direction in which the liquid flows.

  In addition, the separation apparatus according to the present invention can be manufactured using the above-described materials, for example, using a conventional technique such as a sputtering method or a photolithographic method.

  Hereinafter, embodiments of the present invention will be described in more detail with examples. However, the present invention is not limited to the following embodiments.

(Embodiment 1)
An example of the configuration of the apparatus according to the present invention in Embodiment 1 is schematically shown in FIG. The apparatus includes a syringe pump 1 that generates a flow, a flow path 2 that separates particles, a collection tank 3 that collects particles, and a power source 4 that applies a frequency and voltage for performing dielectrophoresis.

  Moreover, the inside seen from the upper surface of the flow path 2 is schematically shown in FIG. The flow path produced by etching the borosilicate glass substrate has a liquid inlet 5 and an outlet 6. In addition, holes of different sizes are formed in the side surfaces of the flow path, and platinum electrodes 8, 9, 10, and 11 for dielectrophoresis are fitted. The upper surface is a glass substrate, and both ends of the flow path are closed by side plates 12. The uneven electrode portion is composed of an uneven electrode (8) and its counter electrode (9), and the capturing electrode portion is composed of a capturing electrode (10) and its counter electrode (11). As shown in FIG. 2, the unevenly-distributed electrode (8) has a smaller electrode area than the counter electrode (9), and similarly, the capture electrode (10) has a smaller electrode area than the counter electrode (11). Is small. Note that the liquid containing particles flows from the inlet 5 to the outlet 6, and the one closer to the inlet 5 is upstream. The power supply 4 has a control system that can supply power to the unevenly distributed electrode part and the capturing electrode part independently. For example, as shown in FIG. 2, a control system that supplies power to the unevenly distributed electrode part; The control system that supplies power to the capturing electrode unit is independent, and has power supply units 15 and 17 that apply independent frequencies and voltages, respectively, and that supplies power to the unevenly distributed electrode unit. And a control circuit 16 for turning on and off the power at predetermined intervals.

  With the above configuration, the unevenly distributed electrode 8 and the counter electrode 9 have different electrode areas, and therefore, when a voltage and a frequency are applied, a non-uniform electric field is generated. Therefore, when particles flow into the unevenly-distributed electrode portion composed of the unevenly-distributed electrode 8 and the counter electrode 9, the particles perform positive dielectrophoresis toward the unevenly-distributed electrode 8. Due to the positive dielectrophoresis of the particles and the flow of the liquid, the particles can be unevenly distributed in the unevenly distributed portion 13. In particular, it is possible to prevent other particles from being captured by the capturing electrode 10 by moving particles (other particles) having a weak dielectrophoretic force away from the capturing electrode 10 provided downstream. In addition, the unevenly distributed electrode portion can flow the unevenly distributed particles downstream of the flow path by turning the power supply on and off at regular intervals.

  Similarly, since the electrode areas of the capturing electrode 10 and the counter electrode 11 are different, a non-uniform electric field is generated, and the particles undergo positive dielectrophoresis toward the capturing electrode 10 having a strong electric field. By causing the particles to be unevenly distributed in the unevenly distributed portion 13 and then flowing to the downstream capturing electrode portion, the capturing portion 14 located on the opposite side of the unevenly distributed portion 13 has a particle having a large dielectrophoretic force (capturing). It moves preferentially from the target particle). Then, particles having low dielectrophoretic force (other particles) are not captured by the capturing unit 14 or are flowed downstream by a fluid action before being captured even if they are attracted to the capturing unit 14 by dielectrophoresis. That is, by causing the particles to be unevenly distributed and then flowing the particles to the capturing electrode portion, the difference in the dielectrophoretic force of the particles can be used effectively, and the separation accuracy can be improved. Note that voltage and frequency are continuously applied to the capturing electrode section.

  A liquid containing particles (for example, an electrolyte solution) is supplied from the inlet 5. The liquid containing the particles not captured by the capturing unit 14 is recovered from the outlet 6 to the recovery tank 3. The collection tank 3 uses a container having a size corresponding to the amount of liquid to be fluidized.

  For example, in the case of an apparatus for separating bacteria and human blood cells, the width of the flow path can be set to 100 μm and the height can be set to 100 μm, for example. The distance between the electrodes A (8) and C (10) embedded in the side surface of the flow path can be about 500 μm, the height (vertical width) can be set to 100 μm, and the width (horizontal width) can be set to 60 μm, for example. The heights of the electrodes B (9) and D (11) can be set to 100 μm, for example, and the width can be set to 280 μm, for example. The above is an example, and it is desirable to select the most appropriate channel width and electrode width according to the dielectric properties of the particles to be captured. It is desirable that the apparatus can apply a voltage range of 10 Vpp to 50 Vpp and a frequency range of 600 kHz to 6.5 MHz. Moreover, the flow rate of a liquid can be set suitably, for example, can be 0.2-1 microliter / s. Note that the height of the flow path can be set as appropriate, and the amount of liquid that can be processed can be increased by increasing the height. For convenience of explanation, the configuration of the apparatus in which the electrode is provided on the side wall of the flow path has been described. However, the configuration is not particularly limited, and for example, the electrode may be provided on the upper and lower wall surfaces.

(Embodiment 2)
The apparatus configuration of the second embodiment is different from the first embodiment only in the configuration of the capturing electrode unit, and is schematically shown in FIGS. 1 and 3. FIG. 3 schematically shows the inside of the flow path 2 as viewed from above. The flow path produced by etching the borosilicate glass substrate has a liquid inlet 5 and an outlet 6. Holes having different sizes are formed on the side surfaces of the flow channel, and platinum electrodes 8, 9, 10, and 11 for dielectrophoresis are inserted to produce the flow channel. The upper surface of the flow path is closed by a glass substrate, and both ends are closed by side plates 12. As shown in FIG. 3, the capturing electrode 10 is composed of a plurality of electrodes, and the total area of the plurality of electrodes is smaller than that of the counter electrode 11.

  In the above configuration, in the plurality of capture electrodes 10 arranged, a non-uniform electric field is formed between each of the capture electrodes 10 and the counter electrode 11, and the flowing particles are directed toward the capture electrode 10 at the shortest distance. Perform dielectrophoresis. Also in this configuration, particles are captured on the surface of each capturing electrode 10 and the capturing section 14 in the vicinity thereof by continuously applying a voltage and a frequency in the capturing electrode section. As in the first embodiment, the electrolyte solution containing the particles is supplied from the inflow port 5, and the liquid containing the particles not held by the capturing unit 14 is recovered from the outflow port 6 to the recovery tank 3. The collection tank uses a container having a size corresponding to the amount of liquid to be fluidized.

  For example, in the case of an apparatus for separating bacteria and human blood cells, the width of the flow path can be 100 μm and the height can be 100 μm. For example, the width of the unevenly distributed electrode 8 and the capturing electrode 10 embedded in the side surface of the flow path is 40 μm, the width of the counter electrode 9 is 180 μm, and the width of the counter electrode 11 is 440 μm. At least two capture electrodes 10 (five in FIG. 3) are arranged, and the distance between adjacent capture electrodes 10 is, for example, 40 μm. The uneven electrode 8 and the counter electrode 9, the capturing electrode 10 and the counter electrode 11 form a counter electrode. The vertical width of each electrode is preferably matched to the height of the flow path. The above is an example, and it is desirable to select the most appropriate channel width, electrode width, flow velocity, frequency, voltage, medium, etc. according to the dielectric properties of the particles to be separated. It is desirable that the apparatus can apply a voltage of at least 10 Vpp to 50 Vpp and a frequency of at least 600 kHz to 6.5 MHz.

(Embodiment 3)
FIG. 4 schematically shows a device configuration for explaining the third embodiment of the present invention. The apparatus includes a syringe pump 18 with a recovery tank that generates a flow and collects particles, a flow path 19 that separates particles, and a power source 20 that applies a frequency and voltage for performing dielectrophoresis.

  FIG. 5 schematically shows the inside of the flow path 19 as viewed from the upper surface. The flow path produced by etching the borosilicate glass substrate has a liquid outflow port 21. Holes having different sizes are formed on the side surfaces of the flow channel, the platinum electrodes 8, 9, 10, and 11 for dielectrophoresis are fitted, and the upper surface is closed with a glass substrate to produce the flow channel. The vertical width of each electrode is the same as the height of the flow path.

  With the above configuration, since the electrode widths of the unevenly-distributed electrode portion including the unevenly-distributed electrode 8 and the counter electrode 9 and the trapping electrode portion including the trapping electrode 10 and the counter electrode 11 are different, a non-uniform electric field is generated between the electrodes. Arise. First, the particles perform positive dielectrophoresis toward the unevenly distributed electrode 8. The particles are unevenly distributed in the uneven distribution portion 13 by the positive dielectrophoresis and the liquid flow, and the particles having a weak dielectrophoretic force in the electrolyte solution are attracted toward the inner wall 7a side of the flow path. Further, in the unevenly distributed electrode section, the power supply is turned on and off at regular intervals to flow the unevenly distributed particles downstream of the flow path. Similarly, a non-uniform electric field is generated in the supplemental electrode section, and the particles undergo positive dielectrophoresis toward the capturing electrode 10. By continuously applying voltage and frequency at the capturing electrode section, particles are captured on the surface of the capturing electrode 10 and the capturing section 14 in the vicinity. The liquid containing particles is supplied from the outflow inlet 21 and circulates in the flow path a plurality of times. After the lap, the liquid containing the particles that are not held by the capturing unit 14 is collected from the outflow port 21 by the pump 18 with a collection tank. The collection tank uses a container having a size corresponding to the amount of liquid to be fluidized.

  For example, in the case of an apparatus for separating bacteria and human blood cells, the width of the flow path can be set to 100 μm, for example, and the height can be set to 100 μm, for example. The width of the unevenly distributed electrode 8 and the capturing electrode 10 embedded in the side surface of the flow path is, for example, 40 μm, and the width of the counter electrodes 9, 11 is, for example, 180 μm. It is desirable to select the most appropriate channel width, electrode width, etc. according to the dielectric properties of the particles to be separated. It is desirable that the apparatus can apply a voltage range of 10 Vpp to 50 Vpp and a frequency range of 600 kHz to 6.5 MHz.

  The following examples of the invention are described. In addition, this invention is not limited to an Example.

<Example 1>
In Example 1, using the separation apparatus described in Embodiment 1 above and a liquid in which human white blood cells K562 and Escherichia coli Eschera coli (E. coli) are mixed, blood cells are captured with an electrode, and Escherichia coli (bacteria) is obtained. Was collected with the liquid.

(Experimental process)
6 (a), (b), and FIG. 7 show that the ubiquitous electrode part and the capture electrode part are arranged in the flow path one by one, and the blood cells (K562) and E. coli are unevenly distributed and captured by dielectrophoresis and flow action. Represents the process to do.

  FIG. 6A shows a state where particles in the flow path are unevenly distributed by the unevenly distributed electrode portion. Blood cells (K562) A and E. coli B gather in the unevenly distributed portion 13 by the non-uniform electric field formed by the unevenly distributed electrode portion. The blood cell (K562) A and E. coli B are unevenly distributed in the unevenly distributed portion 13 on the opposite side of the capture portion 14 across the flow path by dielectrophoresis.

  FIG. 6B shows a state where particles unevenly distributed in the unevenly distributed portion 13 move in the flow direction f and reach the capturing electrode portion. Also in the supplemental electrode part consisting of the capture electrode 10 and the counter electrode 11 located on the downstream side of the unevenly distributed electrode part, the particles undergo dielectrophoresis due to the non-uniform electric field, and blood cells A having a large dielectrophoretic force are captured by the capture part 14. Be captured. On the other hand, Escherichia coli B having a dielectrophoretic force smaller than that of the blood cell A is flowed by a flow action before being captured by the capturing unit 14 and is not captured. Further, the unevenly-distributed electrode portion turns on and off the power supply at regular intervals, thereby separating the unevenly distributed particles from the vicinity of the electrode and allowing the particles to flow downstream. On the other hand, particles are captured by the capturing unit 14 by continuously applying a voltage and a frequency at the capturing electrode unit.

(Experimental conditions)
As the solvent used in this example, a 9.58 w / v% sucrose solution was added with fetal bovine serum (FBS) to make the conductivity 0.056 S / m. To this solvent, blood cells (K562) were added at 10 ⁶ cells / ml and E. coli at 10 ⁸ cells / ml to prepare a liquid containing particles. This liquid was passed through the flow path at a flow rate of 25 μl / min, and dielectrophoresis was performed under conditions of a voltage of 80 Vpp and a frequency of 1 MHz. ON: 10 sec OFF: 2 sec.

  As a result of performing the separation operation under the above conditions, blood cell A was captured by capture unit 14, Escherichia coli B was suspended in the liquid, and blood cell A and E. coli B were separated. The liquid before introduction into the flow path is the initial liquid, the liquid after introduction into the flow path and the liquid after dielectrophoresis is the recovery liquid, and the number of blood cells present in the initial liquid and the recovery liquid is counted using a hemocytometer, and the number of E. coli is counted. Each was measured by the method. From the number of blood cells and E. coli present in the initial solution and the recovery solution, the capture rate to the electrode was determined for blood cells, and the recovery rate to the recovery solution was determined for E. coli. As a result, the blood cell capture rate was 86% and the E. coli recovery rate was 91%. That is, 86% of the blood cells contained in the initial solution was removed, and 91% of E. coli was recovered.

<Example 2>
Using the pump, power source and recovery tank in the separation apparatus described in Embodiment 1, and the flow path described in FIG. 8, blood cells were captured and bacteria were recovered in the process of FIG.

(Experimental process)
FIG. 7 shows a process in which two sets of unevenly distributed electrode portions and capturing electrode portions are arranged, particles are unevenly distributed in the unevenly distributed portion 13 by dielectrophoresis and flow action, and particles are captured by the capturing portion 14.

  Due to the non-uniform electric field derived from the electrode shape, positive dielectrophoresis of the unevenly distributed electrodes 8A and 8B and the capturing electrodes 10A and 10B occurs in each electrode. Each of the unevenly-distributed electrode portions composed of the electrodes 8A and 9A and the electrodes 8B and 9B is turned on and off at regular intervals in the same manner as in Example 1 to separate the unevenly-distributed particles from the vicinity of the electrodes and to the downstream of the flow path. Shed. On the other hand, by continuously applying a voltage and a frequency to each capturing electrode portion composed of the electrodes 10A and 11A and the electrodes 10B and 11B, particles (blood cells) are captured by the capturing portions 14 respectively. By repeating the arrangement of the unevenly distributed electrode part and the capturing electrode part, the blood cell A in the liquid is captured by the capturing part 14, E. coli B floats in the solution, and the blood cell A and E. coli B are separated.

(Experimental conditions)
In this example, as in Example 1, human white blood cells K562 and E. coli E. coli. A liquid in which E. coli was mixed was used. As the solvent, a 9.58 w / v% sucrose solution with FBS added to make the conductivity 0.056 S / m was used. In this solvent, K562 cells were added at 10 ⁶ cells / ml and microorganisms at 10 ⁸ cells / ml. The liquid containing the particles was passed through the flow path at a flow rate of 25 μl / min, and dielectrophoresis was performed under the conditions of a voltage of 80 Vpp and a frequency of 1 MHz. ON: 10 sec OFF: 2 sec.

(Experimental result)
Under the above conditions, blood cell (K562) A was captured by capture unit 14, E. coli B was suspended in the solution, and blood cell A and E. coli B were separated. As a result of obtaining the capture rate for blood cells and the recovery rate for E. coli by the same method as in Example 1, the capture rate for blood cells was 98%, and the recovery rate for E. coli was 91%. That is, 98% of the blood cells contained in the initial solution was removed, and 91% of E. coli was recovered. Increasing the number of unevenly arranged electrode portions and capturing electrode portions increases the capture rate of blood cells, so that it was confirmed that the degree of purification was higher than that in Example 1 when only one set of devices was used. .

<Example 3>
Using the pump, power source and recovery tank in the separation apparatus described in Example 1 and the flow path described in FIG. 10, cells were captured and bacteria were recovered in the process of FIG.

(Experimental process)
FIG. 9 shows a process in which three or more pairs of unevenly distributed electrode portions and capturing electrode portions are arranged, particles are unevenly distributed in the unevenly distributed portion 13 by dielectrophoresis and flow action, and the particles are captured by the capturing portion 14. In the present embodiment, the number of unevenly arranged electrode portions and capturing electrode portions is three.

  In FIG. 10, the electrodes 8A and 9A, the electrodes 8B and 9B, the electrodes 8C and 9C are unevenly distributed electrode portions, the electrodes 10A and 11A, the electrodes 10B and 11B, and the electrodes 10C and 11C are capturing electrode portions. Each is composed.

  Blood cells A and E. coli B are unevenly distributed in the unevenly distributed portion 13 by dielectrophoresis of a non-uniform electric field derived from the shape of the electrode. Due to the fluid action and dielectrophoresis, blood cells A having a strong dielectrophoretic force are captured by the capturing unit 14, and E. coli B having a weaker dielectrophoretic force is caused to flow by the fluid action before being captured by the capturing unit 14. Not captured.

  As in the first embodiment, each unevenly-distributed electrode portion turns on and off the power supply at regular intervals, thereby separating the unevenly distributed particles from the unevenly distributed portion 13 and allowing the particles to flow downstream. On the other hand, particles are captured by the capturing unit 14 by continuously applying a voltage and a frequency at the capturing electrode unit.

  By repeating the arrangement of the unevenly distributed electrode part and the capturing electrode part, the blood cell A is captured by the capturing part 14, E. coli B floats in the liquid, and the blood cell A and E. coli B are separated. By arranging a plurality of unevenly distributed electrode portions and capturing electrode portions, it is considered that the degree of purification is higher than in the first and second embodiments.

(Experimental conditions)
In this example, as in Example 1, human white blood cells K562 and E. coli E. coli. A suspension mixed with E. coli was used. As the solvent, a 9.58 w / v% sucrose solution with FBS added to make the conductivity 0.056 S / m was used. In this solvent, K562 cells were added at 10 ⁶ cells / ml and E. coli at 10 ⁸ cells / ml. This liquid was allowed to flow through the flow path at a flow rate of 25 μl / min, and dielectrophoresis was performed under conditions of a voltage of 80 Vpp and a frequency of 1 MHz.

(Experimental result)
Under the above conditions, blood cell A was captured by capture unit 14, E. coli B was suspended in the liquid, and blood cell A and E. coli B were separated. As a result of obtaining the capture rate for blood cells and the recovery rate for E. coli by the same method as in Example 1, the capture rate for blood cells was 99%, and the recovery rate for E. coli was 91%. That is, 99% of the blood cells contained in the initial solution was removed, and 91% of E. coli was recovered. Since the capture rate of blood cells increases by increasing the number of electrode parts to be arranged, the degree of purification is increased as compared with Example 2 using an apparatus in which two sets of unevenly distributed electrode parts and capture electrode parts are arranged. From the results of Examples 1 to 3, the capture rate is increased according to the number of the unevenly distributed electrode portions and the capture electrode portions. Therefore, it can be said that the degree of purification increases as the number of these electrode portions increases.

It is the schematic which shows an example of the apparatus structure which concerns on this invention. It is the upper surface schematic diagram showing 1st embodiment in the separation apparatus concerning the present invention. It is a top schematic diagram showing a 2nd embodiment in a separation device concerning the present invention. It is the schematic showing an example of the apparatus structure which concerns on this invention. It is a top schematic diagram showing a 3rd embodiment in a separation device concerning the present invention. (A) In the separation apparatus used in Example 1, it is a conceptual diagram which shows the state in which the particle was unevenly distributed by the electrode part for uneven distribution. (B) It is a conceptual diagram which shows the state which caught the particle | grain with the electrode part for a capture | acquisition after unevenly distributing the particle | grains in (a). It is a conceptual diagram which shows the process of arrange | positioning two sets of unevenly-distributed electrode parts and capture | acquisition electrode parts in a flow path, and capturing the particle | grains in a flow path after being unevenly distributed by dielectrophoresis. It is the upper surface schematic of a flow path which shows an example of the structure which has arrange | positioned two sets of the electrode part for uneven distribution, and the electrode part for capture | acquisition in a flow path. It is a conceptual diagram which shows the process which arrange | positions the electrode part for uneven distribution and the electrode part for capture | acquisition in a flow path at least 3 sets, and captures the particle | grains in a flow path after being unevenly distributed by dielectrophoresis. It is the upper surface schematic of a flow path which shows an example of the structure which has arrange | positioned three sets of the electrode part for uneven distribution, and the electrode part for capture | acquisition in a flow path. It is a conceptual diagram which shows the example of the wiring which comprises the electrode for uneven distribution and the electrode for capture | acquisition in the separation apparatus which concerns on this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Pump 2 Flow path 3 Recovery tank 4 Power supply 5 Inlet 6 Outlet 7 Borosilicate glass substrate 8 Uneven electrode 9 Counter electrode 10 Capture electrode 11 Counter electrode 12 Side plate 13 Uneven part 14 Capture part 15, 17 Power supply part 16 Control Apparatus 18 Pump with recovery tank 19 Flow path 20 Power supply 21 Outflow inlet A Particles to be captured (K562 blood cell)
B Other particles (E. coli)
31 Wiring for unevenly distributed electrode part 32 Wiring for capturing electrode part 33 Flow path 34 Flow direction of liquid

Claims (9)

A method in which at least two kinds of particles contained in a liquid flowing in a flow path are dielectrophoresed by a non-uniform electric field generated by an electrode and separated using a difference in dielectric properties,
Flowing the particles downstream from the uneven distribution of the particles by the uneven distribution electrode that generates the first non-uniform electric field;
A trapping target is disposed on the downstream side of the unevenly-distributed electrode, on the opposite side of the particle-distributed side across the flow path, and is configured to generate a second non-uniform electric field. Capturing the particles;
A method for separating particles characterized by comprising:   The uneven distribution electrode and the capturing electrode each attract particles by a positive dielectrophoretic force, and each have an opposing electrode across the channel. 2. The method for separating particles according to 1.   The particle separation method according to claim 1 or 2, wherein particles unevenly distributed in the uneven distribution electrode are caused to flow downstream by turning on and off power to the uneven distribution electrode.   The method for separating particles according to any one of claims 1 to 3, wherein the particles are microorganisms or cells. An apparatus for dielectrophoretic migration of at least two types of particles contained in a liquid and separating them using a difference in dielectric properties,
The device
A flow path for flowing the liquid;
Means for generating a flow action in the flow path;
An unevenly distributed electrode that is installed in the flow path, generates a first non-uniform electric field, and unevenly distributes the particles;
A capturing electrode that is located downstream of the uneven distribution electrode and on the opposite side of the side where the particles are unevenly distributed across the flow path, generates a second non-uniform electric field, and captures the particles to be captured;
A device for separating particles, comprising:   The uneven distribution electrode and the capturing electrode each attract particles by a positive dielectrophoretic force, and each have an opposing electrode across the channel. 5. The apparatus for separating particles according to 5.   7. The particle separation apparatus according to claim 5, wherein the means for generating the fluid action is an injection means, and further has means for recovering the liquid.   The particle separation apparatus according to any one of claims 5 to 7, further comprising means for applying and stopping the first nonuniform electric field to the unevenly distributed electrode at predetermined time intervals.   The particle separation apparatus according to any one of claims 5 to 8, wherein at least two sets of the unevenly-distributing electrode and the capturing electrode are arranged in the flow path.

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