JP5527936B2 - Method and apparatus for separating particles in suspension - Google Patents

Description

  The present invention relates to a method and an apparatus for separating particles to be captured from a suspension containing a particle group composed of at least two kinds of particles using dielectrophoresis.

  Conventionally, when a specific particle is separated from a group of particles composed of a plurality of particles in a suspension by applying a dielectrophoretic force, the frequency is set according to the type of particles to be captured (hereinafter referred to as capture target particles). By selecting, it is possible to apply a positive dielectrophoretic force to the particle group and capture it on the electrode, or to apply a negative dielectrophoretic force to the particle group and exclude it from the electrode. However, for example, in a suspension in which blood cells and microorganisms are mixed, a positive dielectrophoretic force acts on one blood cell and a negative dielectrophoretic force acts on another microorganism. It is impossible to generalize the positive / negative distribution of the dielectrophoretic force for each particle type. Therefore, it is difficult to selectively separate only specific blood cells or microorganisms by applying a positive or negative dielectrophoretic force using dielectric properties unique to blood cells or microorganisms. However, although it is difficult to sort positive and negative, if the electric field is changed at a specific frequency, different dielectrophoretic forces due to differences in polarizability can be applied to blood cells and microorganisms. Depending on the difference, the particles can be separated according to the type.

  In Patent Document 1 or 2, the difference in the dielectrophoretic force of particles flowing at a constant speed is used to separate the particles from the difference in time to reach the outlet.

Moreover, in patent document 3, several types of particle | grains which exist in suspension are once captured by an electrode, and a dielectric characteristic is selectively changed by making an antibody couple | bond. Thereafter, by applying a flow action, only specific particles that specifically react with the antibody are flowed to improve the separation accuracy.
JP 7-505717 A (Patent No. 3182151) JP-A-5-126696 JP 2003-223 A

  However, in the method described in Patent Document 1 or 2, accurate flow rate adjustment is necessary to sort out specific particles. Furthermore, the particles cannot be accurately separated unless the starting position of the liquid to be separated is clearly determined.

  In the method described in Patent Document 3, it is necessary to select a specific antibody. Since selection of a specific antibody is difficult, the separation target is limited.

  As described above, an object of the present invention is a method of separating particles to be captured from a suspension containing a particle group composed of at least two kinds of particles, which does not require flow rate adjustment and does not require treatment of an antibody or the like. It is an object to provide a method and apparatus for capturing and separating specific particles.

The method for separating particles according to the present invention includes:
A method of separating particles to be captured by dielectrophoresis from a suspension containing a particle group consisting of at least two kinds of particles,
An AC voltage between at least two electrodes provided on the substrate by applying by forming a non-uniform electric field, the particles so as to contact with the substrate surface near the between the electrodes the particles by dielectrophoresis Draw
By generating a local flow by the flow generation means provided between the electrodes on the substrate, particles having a low dielectrophoretic force other than the capture target particles are excluded from between the electrodes, and the capture target particles are It captures in the vicinity between the electrodes.

Moreover, the particle separation apparatus according to the present invention comprises:
An apparatus for capturing particles to be captured from a suspension containing a particle group composed of at least two kinds of particles using a difference in dielectrophoresis,
A chamber over to hold the suspension,
Disposed on a substrate constituting the chamber over the non-uniform electric field generated by applying an AC voltage, and at least two electrodes to draw so as to contact with the substrate surface the particles by dielectrophoresis,
A flow generation means provided between the electrodes on the substrate and generating a local flow in the vicinity of the electrodes ; and
Particles with low dielectrophoretic force other than the capture target particles are excluded from the vicinity between the electrodes by the flow, and the capture target particles are captured in the vicinity between the electrodes .

  According to the present invention, by generating a local liquid flow in the vicinity of the electrodes, unnecessary particles attracted in the vicinity of the electrodes can be removed. As a result, it is not necessary to adjust the flow rate, and it is possible to selectively capture specific particles and separate particles in the suspension simply and accurately without using an antibody or the like.

  Since dielectrophoresis is generally well known, a detailed description thereof will be omitted and a brief description will be given. 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 frequency of the applied AC voltage. The force acting on the particles at this time 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.

That is, when a high-frequency AC voltage is applied to the particles, a positive dielectrophoresis in which the particles migrate toward the non-uniform portion where the electric field is strongest due to the action of the AC electric field generated thereby. The dielectrophoretic force F includes the electric field E, the relative dielectric constant ε _{p of} the particle, the relative dielectric constant ε _{m of} the solution containing the particle, the radius a when the particle is regarded as a sphere, the circumference ratio π, the parameter Assuming Re, it is represented by the following equation.

  This equation shows that the force due to dielectrophoresis is affected by the potential gradient, the difference between the relative permittivity of the suspension and the relative permittivity of the particles.

  The particle separation method according to the present invention uses this dielectrophoretic force to draw a group of particles in suspension near at least two electrodes and generate a local flow in the vicinity between the electrodes. By excluding the particle group that approaches or is captured by the flow, only particles having a strong dielectrophoretic force are selectively captured by the electrode and the particles are separated.

Therefore, the method for separating particles according to the present invention comprises:
A method of separating particles to be captured by dielectrophoresis from a suspension containing a particle group consisting of at least two kinds of particles,
An AC voltage is applied between at least two electrodes provided on the substrate to form a non-uniform electric field, the particles are dielectrophoresed, the particles are attracted near the electrodes, and the particles are localized near the electrodes. By generating a dynamic flow,
Particles other than the capture target particles are excluded from the vicinity between the electrodes, and the capture target particles are captured in the vicinity between the electrodes.

(particle)
Particles include cells, intracellular organelles, fungi, actinomycetes, bacteria, viruses or nucleic acids, biological substances such as inorganic substances, crystals or synthetic particles, or liquids or bubbles such as fine water droplets in oil. It is used as a concept that includes a wide range of gases. Although not particularly limited, the biological material is more specifically, for example, nucleotide chain, chromosome, peptide chain, protein, immunoglobulin, serum protein, antibody, antigen, lipid, cultured animal and plant cells, Examples include sperm, nucleic acid, protein, sugar chain, or microorganism. Examples of inorganic substances include oxides such as silica and alumina, metals such as gold, titanium, iron and nickel, polysaccharides such as agarose, cellulose and insoluble dextran, inorganic oxides such as ceramic, polymer latex, polystyrene Alternatively, a polymer compound such as a styrene-butadiene copolymer can be used. The present invention is particularly effective when targeting cells such as blood cells and microorganisms such as bacteria and fungi.

(Suspension)
The suspension in the present invention is a liquid containing a particle group composed of at least two kinds of particles, for example, body fluid such as serum, plasma, spinal fluid, synovial fluid, lymph fluid, urine, fecal matter, etc. Examples thereof include biological samples such as excreta or processed products thereof. As the processed product, for example, a sample obtained by appropriately diluting these biological samples with water or a buffer solution, or by dissolving or suspending particles derived from these biological samples in water or a buffer solution, etc. What was obtained is mentioned.

(electrode)
The electrodes in the present invention are at least two electrodes that form a non-uniform electric field for generating a dielectrophoretic force in a particle group, and are thin-film opposed electrodes that face each other on a substrate. The thin-film counter electrode is a pair of electrodes provided in a thin film on a substrate in a plane-facing manner, and can be formed, for example, by a thin film forming method such as screen printing or photolithography.

  An example of the thin-film counter electrode is a comb-shaped electrode as shown in FIG. The comb-shaped electrode is a counter electrode in which a pair of electrodes having teeth (for example, a width of 30 μm to 100 μm) formed like a comb are inserted and combined in respective grooves (see FIG. 2). When a voltage for dielectrophoresis is applied to a thin-film counter electrode such as a comb-shaped electrode, a non-uniform electric field is formed between the counter electrodes, and the particles are on the substrate side on which the thin-film counter electrode is provided. Will be drawn to. In particular, since the electric field in the vicinity of the electrodes is the strongest, the portion between the electrodes (capturing portion) mainly captures particles. In detail, the edge portion of the electrode has the highest electric field density and a non-uniform electric field. When cells attach to the electrode edge portion, the cell portion becomes a non-uniform electric field. As a result, cells collect between the electrodes.

  Between the electrodes in the present invention, a flow generating means described later is provided, and the flow generating means generates a flow in the capturing part. In the case of a comb-shaped electrode, for example, as shown in FIGS. 1 to 3, the flow generating means can be placed between the electrodes on the substrate. By installing in this way, a flow can be locally generated near between the electrodes.

  The distance between the electrodes can be, for example, 1 μm to 1000 μm, and it is desirable to select the most appropriate value depending on the size of the particles to be captured. When the capture target is bacteria, it is preferably 0.1 μm to 100 μm, and more preferably 1 μm to 10 μm. When the capture target is a cell, it is preferably 10 to 1000 μm, and more preferably 50 to 500 μm.

  The material of the electrode is not particularly limited as long as it is a known material that can be used as an electrode. Examples thereof include metals such as chromium and platinum, conductive materials such as carbon, and transparent electrodes such as ITO. it can. The electrode can be formed by a thin film forming method that combines a screen printing method, a photolithography method, or the like. Specifically, it can be formed by forming a film on a substrate such as a glass substrate or a plastic substrate by sputtering, vapor deposition, plating, or the like, and etching by a photolithography method or the like. The electrode material is preferably a metal with a low ionization tendency that does not cause electrolysis when an alternating voltage is applied.

  For example, when the electrode is a comb-shaped electrode, the thickness of the electrode is preferably 1 μm to 100 μm, and the width of the electrode is preferably 100 μm to 1000 μm.

  The electrodes are connected to a power source that applies an AC voltage for generating dielectrophoresis. The frequency of the alternating voltage is desirably selected as appropriate in consideration of the dielectric constant of the suspension so that the particles to be captured perform positive dielectrophoresis. When the particles to be captured are microorganisms, particularly when the frequency is selected according to the cytoplasmic conductivity, the particles can be collected by applying a positive dielectrophoretic force. Usually, a preliminary examination is performed, and an AC voltage having a single frequency selected optimally is applied between the electrodes. In addition, when the capture target is a cell or a microorganism, the frequency is preferably 1 kHz to 10 MHz, and more preferably 100 kHz to 5 MHz. The induced electrophoretic force received by the particle group is affected by the pH of the suspension, the conductivity, the frequency of the voltage, and the like, so it is desirable to select the separation conditions in consideration of these.

(Flow generation means)
The flow generation means generates a flow locally near the electrodes (particularly between the electrodes). By this flow, it is possible to make it difficult for particles having a weak dielectrophoretic force to approach the vicinity between the electrodes, or to exclude them from the vicinity between the electrodes even if they are attracted.

  As described above, the flow generation means can be provided on the substrate and between the electrodes. In the case where the electrodes are comb-shaped electrodes, for example, as shown in FIGS. 1 to 3, the flow generating means can be placed between the electrodes on the substrate. By installing in this way, a flow can be locally generated near between the electrodes.

  As the flow generation means, any means that can generate convection between the electrodes can be used without particular limitation, and examples thereof include a heating element, a piezoelectric element, a micro pump, and a micro motor. Further, convection may be generated near the electrodes by local heating of the laser. In the case of the heating element, for example, a Peltier element can be arranged between the comb-shaped electrodes. As the piezoelectric element, for example, an element in which a piezoelectric body is sandwiched between two electrodes (piezo element) can be arranged so as to be between the electrodes. In the case of a micro pump and a micro motor, the micro pump and micro motor devices can be arranged on the lower layer side of the dielectrophoresis electrode, similarly to the piezoelectric element. In the case of using the local laser heating means, a configuration in which a laser absorbing portion is disposed between the electrodes, laser irradiation is performed from the outside, and only the electrodes can be locally heated.

  Although it does not specifically limit as a heating element, For example, a Peltier device can be used as a resistor. For example, when the electrodes are comb-shaped electrodes as described above, a Peltier element can be provided between the electrodes and on the substrate (for example, FIG. 1). With this configuration, heat convection can be generated in the capturing unit.

(Embodiment)
Next, an example of a device configuration according to the embodiment of the present invention will be described with reference to FIGS. 1 and 2. The heating part and the electrode part for dielectrophoresis as the flow generating means are formed by laminating a heating part 2, an insulating layer 3 covering the heating part 2, an electrode A4, and an electrode B5 on the substrate 1 (FIG. 1). ). The material of the substrate is not particularly limited, and a known material such as quartz or plastic can be used. In addition, in the apparatus configuration in the present embodiment, the flow generation means is the heating unit, but the present invention is not limited to this.

  The electrode is a thin-film counter electrode having a capturing portion 10 between the electrode A4 and the electrode B5. The thin-film counter electrode is preferably a comb-shaped electrode as shown in FIG. The width of the electrode can be, for example, 10 to 1000 μm. Further, the distance between the electrodes (the distance between the electrode A4 and the electrode B5) can be set to 1 to 1000 μm, for example. The distance between the electrodes, that is, the width of the capturing part 10 can be adjusted as necessary depending on the size of the capturing target particles in the suspension. For example, when the particles to be captured are large such as blood cells, they can be widened (for example, 50 to 500 μm), and when they are small such as bacteria, they can be narrowed (for example, 1 to 10 μm). However, if the distance between the electrodes becomes too wide, the electric field becomes small and the dielectrophoretic force becomes weak, so that it may be difficult to move the particle group or capture the particles to be captured. On the other hand, if it is too narrow, it may be difficult to capture a large amount of particles. Therefore, it is preferable to appropriately adjust the distance between the electrodes in consideration of the characteristics of the particles to be captured and the suspension, the device configuration, and the like. For example, in the case of blood cells (about 10 μm) and bacteria (0.5 to 1 μm) used in the examples of the present invention, the preferred range of the electrode spacing is 100 to 200 μm. Moreover, as a material of an electrode, a metal, carbon, ITO etc. can be used, for example. Furthermore, it can be formed by a thin film forming method combining, for example, a screen printing method or a photolithography method.

  Note that the trapping target particles need not be one type and may be two or more types.

  As the heating unit 2, for example, a thin-film micro heater can be used. And the heating part 2 is arrange | positioned on the board | substrate and between each electrode of the said thin film opposing electrode (namely, board | substrate side surface of the capture | acquisition part 10). The width of the heating unit can be set to 1 to 500 μm, for example, but is preferably adjusted appropriately in consideration of the distance between the electrodes, and is preferably within the distance between the electrodes.

  Moreover, as a material of the heating part 2, a metal, carbon, ITO etc. can be used, for example. Further, like the electrode, it can be formed by a thin film forming method combining, for example, a screen printing method or a photolithography method. The heating unit is not limited to a micro heater, and for example, thermal convection may be generated by local heating using a laser, or convection may be generated by a piezoelectric element or a micro motor.

  The heating unit 2 can be appropriately set to a temperature at which the solution is convected, and the temperature is not particularly limited. Although heat convection can be accelerated by increasing the temperature, the particles may be deformed if the temperature is too high due to the nature of the particles. On the other hand, if the temperature is too low, effective thermal convection may not be obtained. Therefore, it is desirable to set as appropriate in consideration of conditions such as particles, dielectrophoretic force, and frequency. For example, it can be set to 50 to 100 ° C., and is preferably 50 to 70 ° C. for blood cells.

  The insulating layer 3 is not particularly limited as long as it is a material that can electrically insulate the heating element 2 from the electrodes A and B (4, 5), but silicon generally used in the semiconductor field. Examples thereof include inorganic insulators such as oxide films and organic insulators such as resin materials. The insulating layer 3 is not particularly required if there is no need to insulate from the electrode according to the type of flow generating means. When the insulating layer is provided, the thickness of epoxy and polyimide can be about 10 μm (the maximum thickness is 30 μm and the minimum thickness is 1 μm). In the case of silica or alumina, the thickness can be about submicron (2 to 3 μm at the maximum thickness, 5 nm at the minimum thickness).

  The liquid holding unit 11 can be formed by, for example, the spacer 6 and the cover 7 on the substrate on which the heating unit 2 and the electrode unit are arranged, and can have a simple structure. The thickness of the spacer 6 and the cover 7 can be set to 0.1 to 0.5 mm, for example.

  The suspension containing the particle group can be supplied from, for example, the inflow port 8 provided in the cover 7, held by the liquid holding unit 11, and recovered from the outflow port 9.

  By setting it as said apparatus structure, a heat convection can be generated in the capture | acquisition part 10 (namely, vicinity between electrodes) by the heating part 2. FIG. Therefore, it is possible to make it difficult for the particles having a weak dielectrophoretic force to approach the capturing unit 10, and to selectively capture particles to be captured having a strong dielectrophoretic force. Further, once the particles are attracted between the electrodes, heat convection is generated, whereby particles having a weak dielectrophoretic force can be released from the capturing unit 10. Therefore, the capturing target particles can be selectively captured by the capturing unit 10 and the particles can be separated.

  In addition, separation can be performed while flowing the suspension from the inlet to the outlet, and in some cases, circulating the suspension. However, in the present invention, after injecting the suspension into the chamber (liquid holding unit 11), first, the capture target particles are captured in a stationary state, and then a suspension containing particles other than the capture target particles is prepared. It is preferable to collect.

  Furthermore, after capturing the capture target particles in a stationary state, an additional liquid (a liquid medium that does not include a suspension or particle group) is introduced into the chamber from the inflow port 8, and particle groups other than the capture target particles are placed in the chamber. It can also be collected outside. In addition, when the purpose of collection is the particles to be captured, after discharging a particle group other than the particles to be captured out of the chamber, the voltage application between the electrodes can be stopped to recover the liquid containing the particles to be captured. When a particle group other than the capture target particles is discharged out of the chamber, some of the capture target particles may be discharged out of the chamber. It is possible to increase the recovery rate of the particles to be captured by introducing them into the interior and repeating the process.

  In the present invention, first, a plurality of types of particle groups are once attracted to the vicinity of the electrodes by positive dielectrophoresis, and then flow can be generated.

  Here, a method of selectively capturing specific particles, particularly, a particle flow caused by dielectrophoresis and thermal convection will be described in more detail. Although the case of thermal convection will be described, the flow in the present invention is not particularly limited to thermal convection.

  FIG. 3 is a conceptual diagram showing a particle group that undergoes dielectrophoresis and a flow of the particle group due to thermal convection, and shows a state immediately after the occurrence of dielectrophoresis and thermal convection. A high-frequency AC voltage, for example, a DC voltage from the heating unit 2 is applied to the electrode unit. Then, the particles A (12) and the particles B (13) are gathered together in the capturing unit 10 by positive dielectrophoresis. However, since there is a thermal convection generated by the heating unit 2, the particle B (13) having a weak dielectrophoretic force floats due to the thermal convection and moves away from the capturing unit 10. On the other hand, the particles A (12) having a strong dielectrophoretic force do not float due to thermal convection and are captured by the capturing unit 10.

  The AC voltage to be applied is preferably selected as appropriate in consideration of the dielectric properties of the particle group and the suspension. For example, when the particle group is blood cells and bacteria (capture target particles are blood cells), and the conductivity of the suspension is low (for example, 15 mS / m or less), it is 10 Vpp or more and 50 Vpp or less and 100 kHz to 10 MHz. It is preferable.

  FIG. 4 shows a state after a certain amount of time has passed since the occurrence of dielectrophoresis and thermal convection (for example, several seconds later). By continuing the generation of dielectrophoresis and thermal convection, the particles A (12) in the suspension are captured by the capturing unit 10, and the particles B (13) are suspended in the suspension, and the particles A (12) and Particle B (13) is separated.

  In addition, about the liquid containing the particle group discharged | emitted and collect | recovered from the outflow port 9, the density | concentration and kind of particle group, a grasp of a state, etc. can be grasped | ascertained using detection means, such as electricity and light. In addition, when the collected particles to be captured are microorganisms or cells, nucleic acid can be extracted. Furthermore, when the collected particles to be captured are nucleic acids, the nucleic acids collected by the PCR method can be amplified.

  Since the electric field strength decreases when the electrolyte concentration is high, the electrolyte concentration is preferably low. Therefore, particles such as polystyrene are suitable for trapping because the dielectrophoretic force is stronger when the electric field strength is increased by lowering the electrolyte concentration (lowering the conductivity of the solution). On the other hand, in the case of cells and the like, it is necessary to lower the electrolyte concentration for the above reasons, but the salt concentration can be adjusted to some extent to maintain the function. The osmotic pressure can be adjusted with mannitol, for example. In the case of blood cells, although they are in high electrolyte blood, dielectrophoresis is difficult with blood direct, so dilute by the above method. However, considering the dilution efficiency, it is better to be able to use a solution having a slightly higher salt concentration. When the electrolyte concentration is high, it is necessary to increase the voltage in order to increase the electric field strength (since the electric field strength is not uniform, it is also effective to shorten the electrode interval). Also, the optimum frequency changes. However, when the electrolyte concentration is high and the voltage is raised, thermal convection occurs near the electrode. This method separates the force captured by dielectrophoresis and the force of convection. The range of each parameter seems to depend on the electrode configuration (electrode shape and interelectrode distance) and the type of separated particles. In this example (particles are blood cells and bacteria), in the case of this electrode configuration, 30 mS / m or more is required to cause thermal convection, and 50 Vpp at several MHz to obtain dielectrophoretic force for capturing. Above (upper limit is the device limit), it is 150 mS / m or less.

  In the apparatus configuration shown in FIGS. 1 and 2, the method for separating particles in the particle suspension by selective trapping of the particles shown in FIGS. 3 and 4 is generally a low electrolyte that can use dielectrophoresis. The solution is mainly used. That is, when a particle suspension having a low electrical conductivity is used, dielectrophoresis is generated at the electrode section and thermal convection is generated at the heating section 2. However, since a strong electric field is generated between the electrodes during dielectrophoresis, heat is generated between the electrodes depending on the applied voltage and frequency and the electrolyte concentration of the particle suspension. That is, when a solution having a high electrolyte concentration is used, dielectrophoresis and thermal convection may occur simultaneously at the electrode portion. Such a case will be further described below.

  The apparatus configuration in the embodiment of the present invention will be described with reference to FIGS. In the electrode part for dielectrophoresis, an electrode 4 and an electrode 5 are formed on a quartz substrate 1.

  The electrode part is a thin film counter electrode having a capturing part 10 between one pole 4 and the other pole 5. The distance between the electrodes can be set to 1 to 500 μm, for example. The distance between the electrodes, that is, the width of the capturing unit 10 can be adjusted as necessary depending on the size of the particles in the suspension. For example, it can be adjusted according to the particles to be captured so that it is wide for large cells such as blood cells and narrow for small cells such as bacteria. However, if the distance between the electrodes becomes too wide, the electric field becomes small, the dielectrophoretic force is weak, and the generation of thermal convection between the electrodes is also weak. Therefore, the particles can be moved or trapped by dielectrophoresis or suspended by convection It becomes difficult to let you. On the other hand, when it is too narrow, it becomes difficult to capture a large amount of particles. In the case of blood cells and bacteria used in the examples of the present invention, the thickness is more preferably 100 to 200 μm. Moreover, the electrode similar to said embodiment can be used.

  The liquid holding part 11 is formed by a space surrounded by a spacer 6 having a thickness of 0.1 to 0.5 mm and a cover 7 on a substrate on which the electrodes 4 and 5 are arranged, and has a simple structure.

  The particle suspension containing the particles can be supplied from the inlet 8 provided in the cover 7, held by the liquid holding unit 11, and collected from the outlet 9.

  With this device configuration, when dielectrophoresis and thermal convection occur simultaneously at the electrode part, thermal convection is generated from the trapping part where particles gather due to positive dielectrophoresis, and the particles in the particle suspension have weak dielectrophoretic force. Can be removed from the trap. Also, after selectively capturing specific particles, only the removed particles can be recovered. The solution containing the collected particles can be used to identify the concentration and type and to grasp the state by using a detection means such as electricity or light. In addition, when the collected particles are microorganisms or cells, nucleic acid can be extracted. Furthermore, when the recovered particles are nucleic acids, the nucleic acids recovered by the PCR method can be amplified.

  Here, the method for selectively capturing specific particles in the present invention and separating the particles in the particle suspension will be described in more detail. FIG. 7 shows a state immediately after the generation of dielectrophoresis and thermal convection. A high frequency alternating voltage is applied to the electrode part. Both particles A12 and particles B13 gather in the capturing unit 10 by positive dielectrophoresis. The particle B13 having a dielectrophoretic force weaker than that of the particle A12 due to the simultaneously generated thermal convection floats by the convection and moves away from the capturing unit 10. On the other hand, the particles A12 do not float by convection and are captured by the capturing unit 10.

  The AC voltage to be applied depends on the dielectric properties of the particles and the particle suspension. For example, if the particles used in the examples of the present invention are blood cells and bacteria, and the particle suspension is higher in electrolyte than the solution generally used in dielectrophoresis and the conductivity is 30 mS / m or more, 50 Vpp This is 100 kHz to 10 MHz. FIG. 8 shows the state after several seconds after the occurrence of dielectrophoresis and thermal convection. By continuing the generation of dielectrophoresis and thermal convection, the particle A12 in the particle suspension is captured by the capturing unit 10, the particle B13 floats in the suspension, and the particle A12 and the particle B13 are separated.

  Hereinafter, the method for separating particles according to the present invention will be described in detail with reference to Examples. In addition, although the Example shown here is an example of the best embodiment of this invention, this invention is not limited by these Examples.

  The electrode and heating section can be formed by depositing or depositing metal on the substrate and then etching to provide the desired electrode pattern and heating section. A perfusion chamber in which a desired flow path is formed of silicon rubber and glass or polycarbonate is pressure-bonded (adsorbed) onto a substrate on which an electrode and a heating unit are formed.

Example 1
The apparatus configuration used in the present embodiment is as shown in FIGS. 1 and 2, for example, on a substrate 1 such as quartz, a heating unit 2 and an insulating layer 3 as flow generating means, and an electrode A4 for dielectrophoresis. And an electrode B5 (thin film counter electrode) are stacked. The electrode width is 100 μm, and the distance between the electrodes A4 and B5 (electrode distance) is 100 μm. That is, the width of the capturing unit 10 is also 100 μm. The heating unit 2 is a minute heater having a width of 50 μm, and is disposed on the substrate 1 below the capturing unit 10. A Peltier element was used as the minute heater. The minute heater can be a platinum thin film resistor or the like. The spacer is made of silicon rubber having a thickness of 0.5 mm, and the cover 7 is made of glass.

  The suspension is supplied from the inlet 8. Then, after selectively capturing particles to be captured, a liquid containing particles that are not captured by the capturing unit 10 is collected from the outlet 9.

  Next, the particle group and the liquid medium used in the present invention will be described. The particle group used in this example includes human myeloid leukemia cell K562, which is a typical white blood cell, and Escheria. E. coli (E. coli). The liquid medium used was a non-electrolyte sucrose solution generally used in cell dielectrophoresis and fetal bovine serum (FBS) added to adjust the conductivity. In order to obtain an osmotic pressure equivalent to that of blood, the concentration of the sucrose solution was set to 9.58 w / v%. Table 1 shows the relationship between the conductivity, serum concentration, and salt concentration of the adjusted liquid medium.

10 ⁶ cells / ml of K562 cells in these liquid media; E. coli was added at 10 ⁴ cells / ml to prepare a suspension.

  Next, a suspension having an electrical conductivity of 15 mS / m was supplied from the inlet 8 to the liquid holding unit 11, and the suspension was brought into contact with the electrode. Next, an AC voltage of 20 Vpp and 1 MHz were applied to the electrodes, and a DC voltage (5 V) was applied to the heating unit 2. The particles (K562 cells) in the solution were captured by the capturing unit 10 over time. On the other hand, E.I. E. coli was not trapped in the trapping part 10 and floated in the solution. In addition, after supplementing the K562 cells, the K562 cells are removed by recovering the solution from the outlet 9, and the E. coli cells are removed. A solution with floating Coli was obtained. The confirmation was performed by visual observation with a microscope using a hemocytometer.

  In addition, when a 0.5 mS / m suspension was supplied from the inlet, an AC voltage of 10 Vpp, a frequency of 1 MHz was applied to the electrodes, and a DC voltage (5 V) was applied to the heating unit, a suspension of 15 mS / m was obtained. Similarly, K562 cells are removed and E. coli is removed. A solution in which Coli was suspended could be obtained.

  In addition, a suspension of 27 mS / m was supplied from the inlet, and an AC voltage of 30 Vpp, a frequency of 1 MHz was applied to the electrode, and a DC voltage (5 V) was applied to the heating unit, as in the case of the suspension of 15 mS / m. K562 cells were removed and A solution in which Coli was suspended could be obtained.

(Example 2)
The apparatus configuration used in Embodiment 2 of the present invention is that shown in FIGS. In the electrode part for dielectrophoresis, an electrode A4 and an electrode B5 are formed on a quartz substrate 1. The electrode width is 100 μm, and the distance between one electrode A4 and the other electrode B5 is 100 μm. The specimen suspension containing the specimen (particles) is supplied from the inflow port 8 and is surrounded by a silicon rubber spacer 6 having a thickness of 0.5 mm and a glass cover 7 on the substrate on which the electrode portion is arranged. Held in space. After selectively capturing a specific sample, the sample suspension containing the sample that has not been captured by the capturing unit is collected from the outlet 9. With this apparatus configuration, thermal convection is simultaneously generated from the capture unit 10 simultaneously with positive dielectrophoresis, and a sample having a weak dielectrophoretic force in the sample suspension can be removed from the capture unit. Further, after selectively capturing a specific specimen, only the removed specimen can be collected.

  Next, the specimen suspension used in the present invention will be described. The specimen used in this example is the same as that described in Example 1. The liquid medium was prepared by adding fetal bovine serum (FBS) to a non-electrolyte sucrose solution generally used in cell dielectrophoresis and adjusting the conductivity as shown in Table 2 below. Using. In order to obtain an osmotic pressure equivalent to that of blood, the concentration of the sucrose solution was set to 9.58 w / v%. Table 2 shows the relationship between the conductivity, serum concentration, and salt concentration of the adjusted liquid medium.

In these liquid media, 10 ⁶ cells / ml of K562 cells, E. coli. The sample suspension was prepared by adjusting the E. coli to 10 ⁴ cells / ml.

  Next, a method for selectively capturing a specific specimen in the present invention will be described. Specimen suspensions of 56, 110 and 131 mS / m, which have higher conductivity than Example 1, were used. These specimen suspensions were supplied from the inlet 8 and held in a space surrounded by a spacer 6 made of silicon rubber and a glass cover 7 on the substrate on which the electrode portion was arranged. Next, an AC voltage of 50 Vpp and 1 MHz were applied to the electrode part. The K562 cells in the specimen suspension were captured by the capturing unit 10 with the passage of time. On the other hand, E.I. E. coli was not captured by the capture unit 10 but floated in the sample suspension. In addition, after supplementing the K562 cells, the sample suspension is recovered from the outlet 9, whereby the K562 cells are removed from the sample suspension. The sample suspension in which E. coli was suspended could be recovered.

It is sectional drawing which shows an example of the apparatus structure of this invention. It is a perspective view which shows an example of the structure of the thin film counter-type electrode (for example, comb-tooth type electrode) and the flow generation means (for example, heating part) in embodiment of this invention. It is a conceptual diagram for demonstrating the particle | grain separation method of this invention. It is a conceptual diagram for demonstrating the particle | grain separation method of this invention. Sectional drawing which shows the apparatus structure in embodiment of this invention The perspective view which expands and shows the structure of the electrode part in embodiment of this invention The figure for demonstrating the selective capture method of the particle | grains in embodiment of this invention. The figure for demonstrating the selective capture method of the particle | grains in embodiment of this invention.

Explanation of symbols

1 Substrate 2 Heating part 3 Insulating film 4 Electrode A
5 Electrode B
6 Spacer 7 Cover 8 Inlet 9 Outlet 10 Trapping part 11 Liquid holding part 12 Particle A
13 Particle B

Claims (10)

A method of separating particles to be captured by dielectrophoresis from a suspension containing a particle group consisting of at least two kinds of particles,
An AC voltage between at least two electrodes provided on the substrate by applying by forming a non-uniform electric field, the particles so as to contact with the substrate surface near the between the electrodes the particles by dielectrophoresis Draw
By generating a local flow by the flow generation means provided between the electrodes on the substrate, particles having a low dielectrophoretic force other than the capture target particles are excluded from between the electrodes, and the capture target particles are A method for separating particles, wherein the particles are trapped in the vicinity of the electrodes.   The particle separation method according to claim 1, wherein the local flow is generated after the particle group is attracted to the vicinity of the space between the electrodes. Contacting the suspension with the electrode before forming the non-uniform electric field;
Recovering a liquid containing particles other than the capture target particles after removing particles other than the capture target particles from between the electrodes; and
The method for separating particles according to claim 1, wherein:   The particle separation method according to claim 1, wherein the flow is convection by heating.   The particle separation method according to claim 4, wherein the convection is caused by a heating element provided between the electrodes on the substrate. The method for separating particles according to any one of claims 1 to 5 , wherein the particles to be captured are cells or microorganisms.   The method for separating particles according to any one of claims 1 to 5, wherein the particles to be captured are blood cells and are separated from the blood cells and bacteria. An apparatus for capturing particles to be captured from a suspension containing a particle group composed of at least two kinds of particles using a difference in dielectrophoretic force ,
A chamber holding the suspension;
At least two electrodes installed on the substrate constituting the chamber, generating an inhomogeneous electric field by applying an alternating voltage, and attracting the particles to contact the substrate surface by dielectrophoresis;
Provided between the electrodes A on the substrate, we have a, a flow generating means for generating a local flow around between the electrodes,
A particle separation apparatus , wherein particles having low dielectrophoretic force other than the particles to be captured are excluded from the vicinity between the electrodes by the flow, and the particles to be captured are captured in the vicinity between the electrodes . Further, according to claim 8, characterized in that it comprises an inlet for injecting the suspension into the chamber, and outlet for recovering a liquid containing particles other than the acquisition target particles Particle separator. The separation apparatus according to claim 8 or 9 , wherein the flow generating means is a heating element, a piezoelectric element, a micro pump or a micro motor, or a locally heated laser local heating means.