WO2007105784A1 - Dispositif de dielectrophorese - Google Patents

Dispositif de dielectrophorese Download PDF

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Publication number
WO2007105784A1
WO2007105784A1 PCT/JP2007/055169 JP2007055169W WO2007105784A1 WO 2007105784 A1 WO2007105784 A1 WO 2007105784A1 JP 2007055169 W JP2007055169 W JP 2007055169W WO 2007105784 A1 WO2007105784 A1 WO 2007105784A1
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WIPO (PCT)
Prior art keywords
electric field
chip
uniform electric
flow path
fine particles
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Application number
PCT/JP2007/055169
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English (en)
Japanese (ja)
Inventor
Yoshinori Adachi
Fuji Kodera
Kazumasa Horiuchi
Hitoshi Watarai
Original Assignee
Cluster Technology Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Cluster Technology Co., Ltd. filed Critical Cluster Technology Co., Ltd.
Publication of WO2007105784A1 publication Critical patent/WO2007105784A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]

Definitions

  • the present invention relates to an apparatus and method for observing the behavior of a target sample quickly and easily by dielectrophoresis.
  • the sample is polarized by being placed in a non-uniform electric field from the outside, and a dielectrophoretic force is generated to move in a positive or negative direction with respect to the electrode. Therefore, by measuring the behavior of this sample in a non-uniform electric field as the dielectrophoretic mobility, the sample can be characterized or identified. Dielectrophoresis can also be applied to uncharged and large samples. However, for a small sample, the dielectrophoretic force is small and the mobility is small. Therefore, the sensitivity required for analysis could not be obtained, and statistical data had to be obtained using a large amount of samples.
  • This device injects a sample into a chamber containing a multi-electrode array and excites the internal electrodes simultaneously at different frequencies.
  • the multipole array accommodated in this chamber is composed of a series of comb-like spaced electrodes, and the tip of the electrode is placed near the common electrode, so that the injected sample is between each electrode. It is possible to move freely.
  • the particles in the sample solution are preferentially placed on one electrode according to their dielectric properties. It will be gathered.
  • This specific frequency can be used as a characteristic frequency for different particle types, for example, to separate major particles from a solvent by dielectrophoresis. It is also possible to check the homogeneity of a sample purified to a single species. Alternatively, if the injected sample is a mixture, the relative concentration of the mixture of known particles can be determined from data at the appropriate frequency for a certain type of particles present in the mixture and the particle count.
  • This device exceeds the limit of conventional electrophoretic particle separation, that is, separation of particles that cannot be electrophoresed (for example, DNA of 40 kbp or more), Its main purpose is separation, detection, measurement, selection, or separation of substances with the same surface charge, not for particle characterization or identification. In order to characterize the particles using this capillary, it is necessary to repeat measurement at different frequencies, which requires a great deal of labor and time.
  • each device is configured for the purpose of detecting, analyzing, or separating / selecting substances based on the state of fine particles at the end of dielectrophoresis, and the measurement of the behavior of single particles by dielectrophoresis is completed. That can quickly and easily measure sample behavior under different conditions (electric field, electric field strength, applied frequency, sample liquid flow, etc.) at once. is not. Disclosure of the invention
  • An object of the present invention is to provide an apparatus and method for measuring the behavior of fine particles quickly and simply by dielectrophoresis.
  • the present invention provides a microarray dielectrophoresis chip for measuring the behavior of fine particles, the chip comprising:
  • a flow path having a liquid inlet and a liquid outlet
  • a non-uniform electric field generating means capable of forming a non-uniform electric field forming region in at least a part of the flow path, wherein the non-uniform electric field is symmetric with respect to a central axis or a central portion of the non-uniform electric field forming region.
  • the non-uniform electric field generating means is a multipole electrode.
  • the liquid introducing part or the liquid outlet part is an open system.
  • the flow path is a single fly.
  • the direction along the central axis or the flow direction passing through the central portion is a vertical direction.
  • the non-uniform electric field generating means is an electrode capable of increasing or decreasing the electric field intensity along the central axis or along the flow direction passing through the central portion.
  • a part of the non-uniform electric field generating means is exposed in the flow path.
  • the surface of the non-uniform electric field generating means exposed in the flow path protrudes from the wall that supports the non-uniform electric field generating means.
  • the non-uniform electric field generating means includes an electric field generating unit having a curved surface according to the Laplace equation, and an auxiliary unit.
  • the length of the electric field generating part of the non-uniform electric field generating means is longer than the length of the fine particles.
  • the entire inner surface of the channel is coated with a hydrophilic membrane or a biocompatible membrane.
  • the liquid introduction part is provided with a liquid reservoir.
  • the chip substrate is made of an opaque material.
  • a measurement window for measuring fine particles present in the non-uniform electric field forming region is disposed at an end of the flow path.
  • a part of the non-uniform electric field generating means includes a wire extending in a direction opposite to the flow path, and the wire is connected to a power source.
  • the present invention also provides a method for producing any one of the above chips, the method comprising obtaining a chip substrate having holes for disposing the non-uniform electric field generating means and holes to be flow paths;
  • a resin thread or a pin is used as a jig in the step of fitting the non-uniform electric field generating means.
  • the present invention also provides another manufacturing method of any of the above chips, Is
  • the present invention further provides a further method for producing any of the above chips, the method comprising:
  • the present invention further provides a microarray dielectrophoresis device for measuring the behavior of fine particles, the dielectrophoresis device comprising:
  • Means for measuring the position of the fine particles in the non-uniform electric field forming region of the chip are provided.
  • the apparatus further comprises means for injecting a sample containing fine particles and a liquid for electrophoresis into the channel of the cup.
  • the injection means is an inkjet device.
  • an electrophoretic liquid drive means is further provided.
  • the chip, the driving means, and the communication path between the liquid introduction part of the chip and the driving means are arranged at the same height.
  • the driving means is an ink jet device.
  • the apparatus further comprises means for calculating dielectrophoretic mobility from the measured position of the fine particles.
  • it further comprises means for stimulating the microparticles.
  • the energization means can increase or decrease the electric field intensity so that the fine particles can migrate in the direction of the central axis of the flow path of the chip.
  • the energization means can change the electric field strength immediately near the electrode in a direction different from the direction of the electrode, thereby preventing the sample from attaching to the electrode.
  • the present invention also provides a method for measuring the behavior of microparticles, the method comprising introducing a migration liquid into the flow path of any one of the microarray dielectrophoresis chips described above;
  • the focus of the incident light from the means for measuring the position of the fine particles in the field forming region is moved in the direction along the central axis of the non-uniform electric field forming region of the flow path or in the flow direction passing through the central portion.
  • a flow is generated in the electrophoresis liquid.
  • the electrophoresis liquid is degassed.
  • the liquid introduction part to the flow path is an open system and a liquid reservoir is provided in the liquid introduction part
  • the electrophoretic liquid is supplied to the liquid reservoir so that the liquid level becomes convex.
  • the diameter of the incident light to the non-uniform electric field forming region is larger than the diameter that interferes with differential interference or phase interference in the flow path.
  • the incident light to the non-uniform electric field forming region is irradiated so as not to hit the wall of the flow path.
  • the light incident on the non-uniform electric field forming region is straight light or convergent light.
  • the diameter of the incident light to the non-uniform electric field forming region is set to be larger than a diameter that interferes with differential interference or phase interference in the flow path.
  • the behavior of fine particles in different electric fields can be measured quickly and simply as the dielectrophoretic mobility. Can do. Using this effort's chip or device, it is possible to analyze multiple individual particles without the need for large samples. Yes, the analysis speed is fast. Furthermore, if a database is created, it will be possible to identify particles and to estimate (discriminate) substances. In addition, since there is no damage due to the addition of marker substances or contact, the sample used for analysis can be reused, and the fine particles used for analysis can be used for further precision analysis.
  • the chip of the present invention having a flow path and a non-uniform electric field generating means may be used in combination with a commercially available observation apparatus (such as an optical microscope) for simple analysis. it can.
  • a commercially available observation apparatus such as an optical microscope
  • a plurality of channels are easily provided in a single chip, a plurality of samples can be measured simultaneously under the same conditions or a single sample can be measured simultaneously under different conditions. Therefore, it can be applied to quality control.
  • FIG. 1 is a top sectional view schematically showing the configuration of the dielectrophoresis chip of the present invention.
  • FIG. 2 is a cross-sectional view schematically showing the configuration of the dielectrophoresis chip of the present invention.
  • FIG. 3 is a longitudinal sectional view and a transverse sectional view schematically showing the structure of the electrode of the chip for dielectrophoresis of the present invention.
  • Fig. 4 is a graph showing an example of the distribution of electric field and the behavior of fine particles in a cross-section of the tip of the dielectrophoresis chip of the present invention.
  • B is ⁇ 1 5
  • O / m The case of O / m is shown.
  • FIG. 5 is a cross-sectional view schematically showing an example of the configuration of the dielectrophoresis chip of the present invention.
  • FIG. 6 is a conceptual diagram schematically showing the configuration of the dielectrophoresis apparatus of the present invention.
  • FIG. 7 is a graph showing the change over time of the travel distance of polystyrene standard spheres of ⁇ 1 z m and ⁇ 5 ⁇ at an applied frequency of 100 kHz.
  • FIG. 8 is a graph showing the change over time of the travel distance of the polystyrene standard spheres of ⁇ 1 ⁇ and ⁇ 5 ⁇ for the applied frequency of 2 ⁇ .
  • FIG. 9 is a graph showing the relationship between applied frequency and dielectrophoretic mobility in polystyrene standard spheres of ⁇ 1 ⁇ and ⁇ 5 ⁇ m.
  • FIG. 10 is a graph showing the relationship between applied frequency and dielectrophoretic mobility for yogurt and yeast. BEST MODE FOR CARRYING OUT THE INVENTION
  • fine particles refers to various fine particles such as inorganic fine particles, organic fine particles, and biological fine particles having a size of about 1 nm to about 1 mm.
  • examples of such fine particles include inorganic metal oxides such as silica and alumina; metals such as gold, titanium, iron and nickel; inorganic metal oxides into which functional groups have been introduced by an operation such as silane coupling treatment; Polysaccharides such as agarose, cellulose, insoluble dextran; polymer particles such as polystyrene latex, styrene / butadiene copolymer, styrene / methacrylic acid copolymer, acrolein / ethylene glycol dimetatalylate copolymer; microorganism (yeast, bacteria , Viruses), cells (red blood cells, white blood cells, virus-infected cells, etc.), sugars, nucleic acids (DNA, RNA, etc.), proteins (enzymes, etc.
  • non-biological particles such as latex beads may be bound to or covered with biological materials such as microorganisms, cells, viruses, plasmids, or chemically active species.
  • the biological microparticle may be a body fluid such as serum, plasma, cerebrospinal fluid, synovial fluid, lymph fluid, or a processed product of a biological sample such as excrement such as urine and feces.
  • a treated product is preferably diluted, dissolved or suspended as appropriate with water or a buffer solution.
  • Biological fine particles include those chemically synthesized. .
  • “behavior of fine particles” means that fine particles arranged mainly in a non-uniform electric field move due to dielectrophoretic force, and the dielectrophoretic mobility of fine particles is used as an indicator of this behavior.
  • the dielectrophoretic mobility of fine particles is Represented by in the formula:
  • lo gR t lo gR 0 + at
  • R t the distance from the center after t seconds is represented by R t (see Figure 1).
  • the distance is preferably a plane perpendicular to the flow direction passing through the central axis or central portion of the non-uniform electric field forming region formed in at least a part of the flow path, that is, a non-uniform electric field surface having an arbitrary electric field strength.
  • the horizontal distance at. In some cases, the movement of particles due to sample sedimentation, solution flow, etc., is measured as the behavior of fine particles.
  • the dielectrophoretic force F DEP > is expressed by the following formula:
  • the dielectrophoretic mobility ⁇ since the dielectrophoretic mobility ⁇ ; has no position dependency, it can be simply evaluated only at the electrophoretic start position and the end position.
  • the target sample In dielectrophoresis, the target sample must be suspended.
  • the electric field When floating with dielectrophoretic force, the electric field is not uniform in the vertical direction (z-axis direction), so the electrophoretic force varies depending on the z-axis position.
  • measurement when floating by balancing with an external force such as gravity, measurement is possible only when the balance is well balanced. If the distribution of the inhomogeneous electric field is unknown, sufficient linear parameters for measurement cannot be obtained.
  • the chip for microarray dielectrophoresis of the present invention is a non-uniform electric field generating means capable of forming a non-uniform electric field forming region in at least a part of a flow path having a liquid introduction part and a liquid lead-out part.
  • the central axis or the non-uniform electric field forming region Forms a non-uniform electric field so as to be symmetric with respect to the central part, and forms a uniform electric field in the direction along the central axis or in the flow direction through the central part, or along the central axis or the Means for increasing or decreasing the electric field strength or forming different electric fields along the flow direction through the central portion.
  • Such a chip of the present invention forms a non-uniform electric field so as to be symmetric with respect to the central axis or central part of the non-uniform electric field forming region, and in the direction along the central axis or in the flow direction passing through the central part.
  • Average the electric field strength can be increased or decreased or different electric fields can be formed along the central axis or along the direction of flow through the central part. It is possible to measure the behavior (dielectrophoretic mobility) of fine particles in a single shape.
  • the “non-uniform electric field forming region” is a region where at least a part of the flow path can be formed by the non-uniform electric field generating means provided on the chip.
  • the chip of the present invention has at least a pair of electrodes as a non-uniform electric field generating means, for example, inside or outside a flow path such as one Is provided.
  • the base material of the chip is not particularly limited as long as it is an insulating material (non-conductive material). In order to prevent scattering of incident light during measurement, opaque materials are preferred. Examples of such a material include a silicone resin, or a resin obtained by mixing a coloring material with an epoxy resin, an acrylic resin, a phenol resin, or the like. Alternatively, if the chip itself is relatively transparent, a light blocking object may be placed above or below the chip.
  • the chip of the present invention is provided with a plurality of flow paths.
  • the direction along the central axis of the non-uniform electric field forming region or the flow direction passing through the central portion is preferably perpendicular to the chip surface, that is, the vertical direction.
  • the diameter is usually from 100 nm to 5 mm, preferably from 10 ⁇ m to 1 mm, and more preferably from 10 ⁇ to 500 ⁇ m.
  • the diameter is usually from 100 nm to 5 mm, preferably from 10 ⁇ m to 1 mm, and more preferably from 10 ⁇ to 500 ⁇ m.
  • the non-uniform electric field forming region is usually composed of a chip base material, and the material is preferably an insulating substance (non-conductive substance).
  • the length of the non-uniform electric field forming region is usually about 0.1 to 5 mm, more preferably 0.1 mm to 2 mm, and still more preferably 0.5 mm to 1 mm.
  • the entire inner surface of the flow path is covered with a hydrophilic film or a biocompatible film.
  • the shape of the inner surface of the flow path may be such that the electrophoresis liquid has a flow rate having a profile. For example, if the cross section is circular, the flow velocity in the center is fast and the flow velocity near the wall of the flow path is slow.
  • the liquid introduction part of the flow path may be either an open system or a closed system.
  • An open system is preferred because the solvent in the flow path can be easily replaced or replenished, and because an ink jet device can be used as a means for forcibly injecting the sample.
  • the sample can be injected from the top or from the bottom through the tube.
  • liquid introduction part of the flow path is an open system
  • a liquid reservoir is provided in the liquid introduction part.
  • the liquid volume does not change when a small amount is sucked with the pump, and the infusion rate can be stabilized accordingly.
  • an introduction path that continues from the liquid reservoir may be provided so as to continue to the flow path.
  • the liquid reservoir is symmetrical so that the light incident on the flow path during measurement can be made symmetrical with respect to the non-uniform electric field forming region by adjusting the upper part of the liquid level in the liquid reservoir to a horizontal, convex, or concave surface.
  • the liquid holding structure has a shape (for example, a sufficiently large cylindrical shape with respect to the flow path).
  • a shape for example, a sufficiently large cylindrical shape with respect to the flow path.
  • the diameter of the light incident hole at the time of measurement is too large to interfere with differential interference or phase interference in the flow path.
  • a measurement window such as an acrylic plate shown in FIG. 2 may be provided at the end of the flow path.
  • a material having good light transmittance is used, and examples thereof include acrylic resin and quartz.
  • the chip of the present invention includes non-uniform electric field generating means.
  • An example of the non-uniform electric field generating means is an electrode.
  • the shape of the electrode is not particularly limited as long as it can form a spatially nonuniform electric field in the nonuniform electric field forming region.
  • the size of the electrode itself is not particularly limited as long as it has an electrode structure in which the sample that receives the negative dielectrophoretic force gathers and the electrode does not exist in the vertical direction.
  • the inner diameter of the non-uniform electric field forming region for example, the first one
  • it may be thick or thin like a wire.
  • the number of electrodes is not limited to a quadrupole, and may be a double pole or an octupole.
  • each electrode is preferably formed such that the boundary of the electrode cross-section is represented by a function f (x, y) that satisfies the Labrass equation.
  • f (x, y) is, a (x 2 -y 2) + bxy, a (x 3 - 3 xy 2) + b (y 3 - 3 x 2 y ), and a - is represented by (x 4 6 x 2 y 2 + y 4) + b (x 3 y- xy 3).
  • a and b are constants.
  • the shape of the electric field application surface in the non-uniform electric field forming region is preferably a columnar shape, a truncated cone shape, a trumpet shape, or the like.
  • the electrodes are arranged rotationally symmetrically, creating a cross buil and enabling more precise analysis.
  • the electrode used in the chip of the present invention is made of, for example, a conductive material such as carbon or a noble metal, and its structure has a dielectrophoretic force in one direction of the mouth passing through the central axis or the center of at least a part of the flow path.
  • Electrode materials include amorphous carbon, carbon, gold, platinum, super steel, and zinc alloys.
  • the electrode diameter varies depending on the particulate to be analyzed. Usually, the diameter is 100 nm to 5 mm, preferably 10 ⁇ m to 1 mm.
  • the distance between the electrodes depends on the precision of microfabrication, and is usually 500 ni or less, 0.1 m or more, and preferably 75 m or less and 1 tm or more.
  • the diameter and spacing of the multipole electrodes placed in each channel can be measured, for example, biological particles such as viruses, prions, proteins, DNA, and chemically active particles such as coated latex beads. It is also possible to change according to the target fine particles. If the distance between the electrodes is extremely large, it is not possible to form a nonuniform electric field with sufficient electric field strength to analyze the fine particles to be measured without causing bubbles or temperature changes due to electrolysis or heating.
  • the length of the electric field generating part of the non-uniform electric field generating means (that is, the electrode length) is not sufficiently large with respect to the fine particles to be measured, a uniform electric field cannot be applied to the fine particles to be measured. Can't get.
  • the distance from the central axis or the central portion of the non-uniform electric field forming region is different.
  • the electrode is preferably composed of an electric field generating unit having a curved surface according to the Laplace equation as described above and an auxiliary unit (see FIG. 3).
  • the role of the auxiliary part is to increase the electrode cross section.
  • the electrical resistance of the electrode itself decreases; wiring becomes simple (wiring is difficult if it is small); wiring can be thickened, so the resistance of the wiring decreases;
  • Conductive material forms non-uniform electric field by moving away from non-uniform electric field formation region Prevents intrusion into the area; prevents short-circuiting between electrodes because it is not necessary to perform wiring in a narrow space between electrodes; makes electrode creation easy; makes handling of electrodes easy; positioning The taper of the auxiliary part makes it easy to release during molding.
  • the exposed surface of the electrode in the flow channel protrudes from the wall (wall forming the capillary) supporting the electrode to the central axis of the flow channel.
  • the wall wall forming the capillary
  • the exposed surface of the electrode in the flow channel protrudes from the wall (wall forming the capillary) supporting the electrode to the central axis of the flow channel.
  • FIG. 4 shows an example of the electric field distribution of IEI 2 and the behavior of fine particles of the chip for dielectrophoresis of the present invention.
  • the unit of numerical values in the figure is an arbitrary unit. Electrodes are arranged at the four corners of A and B in Fig. 4, and the electric field is distributed concentrically in the flow path. In this example, when the wall of the channel is located farther from the center than the electrode, the electric field is concentrically shaped around the electrode.
  • the fine particles near the center of the flow path move outward according to the density of the contour lines of the electric field, for example, and the fine particles near the electrode Due to the distorted electric field density, it moves in the direction between adjacent electrodes without adhering to the electrodes.
  • the electrode surface is covered with a dielectric, the electric field strength decreases accordingly, so that a part of the electric field generating part of the non-uniform electric field generating means is exposed in the flow path. It is preferable.
  • the wall surface of the flow path between the electrodes draws a smooth curved surface in that it can prevent turbulent flow and generate laminar flow when flow occurs in the flow path. It is preferable.
  • the surface of the portion exposed to the flow path may be covered with a hydrophilic film or a biocompatible film. Examples of such membranes include phospholipid membranes, and examples of materials include PDMS (polydimethylsiloxane).
  • the non-uniform electric field generating means provided in the chip of the present invention forms a non-uniform electric field so as to be symmetric with respect to the central axis or central portion of the non-uniform electric field forming region, and the direction along the central axis or the A uniform electric field is formed in the flow direction passing through the central part, or the electric field strength is increased or decreased along the central axis or along the flow direction passing through the central part.
  • the electrophoretic force in the direction of the central axis can be reduced by sufficiently increasing the increase / decrease in the electric field strength in the plane perpendicular to the central axis, relative to the increase / decrease in the electric field strength in the central axis direction.
  • the change in the electric field strength E r on the plane perpendicular to the central axis with respect to the change in the electric field strength E z in the direction of the central axis is VE r>> VE z
  • VE r>> VE z This allows the dielectrophoretic force in the direction of the central axis to be negligible with respect to the vertical direction, and the direction of the dielectrophoretic force can be approximated only to the direction perpendicular to the central axis, and can be processed as a scalar instead of a tensor. And This makes it possible to conduct dielectrophoresis experiments with multiple intensities in a single location.
  • the distance between the electrodes is changed along the flow direction passing through the central axis or center of the non-uniform electric field forming region; Changing the voltage applied to each part of the electrode along the central axis by using the material; weakening the electric field by coating the surface of the electrode with an insulating or dielectric material; dividing the electrode along the central axis Changing the voltage applied to each electrode; placing an electrode that creates an opposite electric field behind the electrode on the opposite side of the flow path; For example, the applied voltage may be changed as the sample moves.
  • each electrode is formed in a truncated cone shape and arranged in parallel. These electrodes may be inclined and arranged radially. For example, length 5 0 0 ⁇ ! Up to 1 mm electrode vertically along the fly
  • the electric field shape When placed in parallel to the Z axis, the electric field shape does not change and the dielectrophoretic force does not change even if the sample moves in the z axis direction.
  • a part of the non-uniform electric field generating means may be provided with a wire extending in the direction opposite to the flow path (see FIG. 5). This wire can be connected to an external power source for driving dielectrophoresis.
  • a connector for connecting the communication path between the flow path and the liquid drive means to the flow path may be provided at the lower portion of the chip.
  • An example of this connector is rubber.
  • the method for manufacturing the above chip is not particularly limited. However, a chip capable of more accurate measurement can be manufactured by any of the chip manufacturing methods of the present invention.
  • the first method of the present invention comprises a step of obtaining a chip substrate having holes for arranging the non-uniform electric field generating means and holes to serve as flow paths; and a hole for arranging the non-uniform electric field generating means. Fitting the non-uniform electric field generating means to the step.
  • the “hole to be a flow path” penetrates the chip base material, but the “hole for arranging the non-uniform electric field generating means” may penetrate as necessary and penetrates. It does not have to be.
  • a hole may be formed in a chip substrate of an appropriate size, for example, by using a tool, a laser or the like.
  • a substrate may be manufactured.
  • the electrode is sealed using resin. It is preferable to use an elastic resin thread or a pin having a depression along the curved surface of the electrode as a jig for positioning the electrode during electrode placement. Furthermore, when sealing, sealing with the same shape as the curved surface of the electrode can prevent the sealing resin and the adhesive from adhering to the electrode surface. By using a conductive adhesive, the occurrence of electric field distortion can be prevented.
  • the second method of the present invention is a step of obtaining a plurality of chip base materials that can form a flow path by combining them, wherein each of the chip base materials has a direction or central portion along the central axis. A portion of the flow path that is divided in a flow direction passing therethrough; a step of disposing a non-uniform electric field generating means on each chip substrate; and a plurality of the non-uniform electric field generating means Combining the chip substrates.
  • the third method of the present invention is a step of obtaining a plurality of chip base materials that can form a flow path by combining them, wherein each of the chip base materials has a direction or central portion along the central axis. Including a part of the flow path divided in a flow direction passing therethrough; and a step of arranging the non-uniform electric field generating means at the same time as combining the respective chip base materials.
  • the flow path is formed by combining a plurality of chip base materials that form the walls of the flow path.
  • the flow path is a part of the wall of the flow path divided in the direction along the central axis or in the flow direction passing through the central portion.
  • a part of the flow path may be formed on a chip substrate having a size by using a tool, a laser, or the like.
  • the chip substrate may be manufactured by molding with a mold.
  • the step of disposing the non-uniform electric field generating means on the chip substrate is performed by previously forming a part for disposing the non-uniform electric field generating means on the chip base by using, for example, a tool, a laser, etc. In this way, non-uniform electric field generating means may be fitted.
  • the microarray dielectrophoresis apparatus includes a microarray dielectric swimming chip according to the present invention, a stage for disposing the chip; a means for energizing the nonuniform electric field generating means of the chip; and a nonuniform electric field forming region of the chip Means for measuring the position of the fine particles therein.
  • the stage for disposing the chip includes at least a sealing unit, a fixing unit, and a tube that communicates the flow path and the liquid driving unit.
  • the microarray dielectrophoresis apparatus of the present invention may include liquid driving means (for example, a syringe pump or an ink jet device) connected to such a tube. If the liquid driving means is used, for example, the sample is placed in a liquid reservoir provided on the inlet side of the flow channel, and the sample is introduced into the flow channel by sucking with a pump or the like from the outlet side of the flow channel. Can do.
  • the chip, the driving means, and the communication path between the liquid introducing portion of the chip and the driving means are arranged to have the same height.
  • a guide groove may be provided on the stage so that the tube connected to the acrylic flow path does not interfere with other equipment.
  • the syringe pump may be equipped with a three-way valve, It may be possible to automatically switch between capturing and driving the fluid. Inserting a filter in the flow path can also prevent measurement samples and dust from entering the flow meter and pump.
  • the sealing means is a sealing means usually used in the technical field such as a sealing rubber, and examples thereof include silicone rubber.
  • the apparatus of the present invention may have a measurement window similar to that provided on the above-mentioned chip on the stage.
  • the fixing means is not particularly limited, and may be screwed or may be clamped on the stage with a clip or the like.
  • a screw hole is provided on the stage.
  • the electrode tip is applied by applying pressure from above using an electrode fixture, and more preferably using the means for fixing and energizing the electrode (the same as the means for energizing the non-uniform electric field generating means). Fix it. At this time, the pressure on each electrode should be equal.
  • the means for energizing the non-uniform electric field generating means is not particularly limited. It is preferable that an arbitrary voltage change, frequency change, and the like are possible for each electrode.
  • the apparatus of the present invention may include an injection means for introducing the sample and the liquid for electrophoresis into the flow path.
  • the injection means may be any means as long as it can inject the sample and the liquid for electrophoresis into the flow path, and an ink jet device is preferred.
  • the ejection port of the inkjet device is arranged at the upper part (introduction part) of the flow path inlet, and the position thereof is along the central axis of the non-uniform electric field forming region or the flow direction passing through the central part. Can be adjusted to inject the sample. .
  • the apparatus of the present invention comprises means for measuring the distance (ie, position) from the center of the fine particles in the non-uniform electric field forming region of the flow path and the position change.
  • Any measuring means may be used as long as it can detect fine particles in the non-uniform electric field forming region.
  • Examples include a load binding device (CCD) camera, a confocal laser microscope, and a fluorescence microscope. In both cases, fine particles are visualized by the incident light from the measurement window and can be detected.
  • CCD observation means observation by visible light, fluorescent light, phase interference, differential interference, etc. is possible.
  • Such detection devices are arranged on the side of the chip where the measurement window is provided, and the inside of the non-uniform electric field forming region is observed from the measurement window.
  • the apparatus of the present invention may further comprise means for calculating dielectrophoretic mobility from the measured positions of the fine particles.
  • the measured position change can be stored together with the frequency applied to the non-uniform electric field forming region, for example, in a computer.
  • the computer calculates the dielectrophoretic mobility from the measured position based on the above formula.
  • means may be provided for spectralizing as a function of the high frequency electric field frequency to generate a non-uniform electric field.
  • it is necessary to use data having various frequencies. Therefore, it is preferable to use an apparatus provided with a plurality of non-uniform electric field forming regions capable of measuring position changes at different frequencies at the same time.
  • the obtained data can be output as a dielectrophoretic mobility spectrum showing frequency dependence.
  • the dielectrophoretic mobility of the fine particles can be reduced.
  • the frequency spectrum shown can be obtained. Or the data acquired for various particles It can also be integrated by computer and made into a database.
  • the solution is flowed in the flow direction along or passing through the central axis of the flow path to create a flow velocity distribution, and the combined effect of movement by dielectrophoresis and the velocity of the sample flowing in these directions is generated. It is also possible to perform more precise measurements.
  • the electric field intensity varies along the central axis of the flow path or along the flow direction passing through the central portion, so that the trajectory of the behavior of the fine particles at various electric field strengths can be recorded.
  • the obtained dielectrophoretic mobility or the spectrum or trajectory is used to calculate the dielectrophoresis of the known particle in this database using a computer.
  • Fine particles can be identified by collating with mobility or frequency spectrum or trajectory.
  • the apparatus of the present invention may include means for such verification.
  • the apparatus of the present invention may further include means for stimulating the fine particles.
  • a stimulus means an electrical, physical, or drug stimulus.
  • Means for giving such stimulation include, for example, means for dropping a drug onto a sample containing fine particles by a pipette ink jet device or the like; means for arranging a sample containing fine particles in the stimulation device; A means for inserting a terminal or the like into a container containing a sample containing, a means for inserting a drug dropping pipe, an electrode, or an antenna into a channel system or a wall surface.
  • electromagnetic stimulation or impact can be given by irradiation from outside the chip.
  • the apparatus of the present invention may further include a means for separating and collecting fine particles based on the calculated dielectrophoretic mobility.
  • a fractionation device that can collect and collect discharged samples, and deliver discharged samples to other devices. Examples include connected tubes.
  • the method of the present invention for measuring the behavior of microparticles using such a dielectrophoresis apparatus comprises the step of introducing a migration liquid into the flow path of the microarray dielectrophoresis chip of the present invention; The position R of the fine particles introduced into the non-uniform electric field forming region.
  • calculating a dielectrophoretic mobility from R t Specifically, for example, the measurement can be performed by the following procedure.
  • the electrophoresis liquid is preferable as the electrophoresis liquid, but is not limited to this, and various organic or inorganic solvents can be used.
  • the electrophoretic liquid may be used as it is, and it is preferable to adjust the conductivity, density, osmotic pressure, pH, etc. in relation to the fine particles. However, the addition of substances that interfere with the AC electric field is not preferred.
  • the electrophoresis liquid is preferably degassed in advance.
  • the sample containing the fine particles to be measured is injected into the flow path or the upper liquid reservoir or the lower liquid introduction part previously filled with the electrophoresis liquid.
  • the liquid for electrophoresis may be supplied to the liquid reservoir in consideration of incident light so that the liquid surface of the liquid reservoir becomes convex.
  • the fine particles are moved to a predetermined position in the non-uniform electric field forming region provided with the non-uniform electric field generating means such as an electrode by causing the liquid drive means such as a pump to cause flow or settling.
  • a sample placed at a predetermined position is observed with a means for measuring the above position, for example, a CCD camera, a laser, an electromagnetic measurement means, etc., and stored in a computer.
  • a means for measuring the above position for example, a CCD camera, a laser, an electromagnetic measurement means, etc., and stored in a computer.
  • an electric field (electric field) is applied to the stationary sample.
  • the strength of the electric field formed inside is usually 3.5 MV / m or less, preferably 1.0 MV / m or less.
  • the applied frequency is usually 1 OH z to: L 00 MHz, more preferably 100 Hz to 10 MHz.
  • the electric field formed in the present invention may be either a DC electric field or an AC electric field, but is preferably an AC electric field.
  • the electrode portion may be appropriately cooled.
  • the AC voltage to be applied is usually 2 to 90 V, preferably:! ⁇ 20V, and the frequency is usually 10Hz ⁇ : 100MHz, preferably 10OHz ⁇ 10MHz. If necessary, the AC electric field strength or frequency may be changed during electrophoresis.
  • Electrophoretic force can be reduced. Since the dielectrophoretic force is proportional to the change in the electric field strength, the change in the electric field strength E r in a plane perpendicular to the central axis with respect to the change in the electric field strength E Z in the central axis direction is expressed as VE r >> VE z.
  • the behavior of the fine particles in the sample generated by the formed electric field is observed by the above means and stored in a computer.
  • a flow is generated in the electrophoresis liquid, and the focus of the incident light from the means for measuring the position of the fine particles in the non-uniform electric field forming region according to the movement of the fine particles by the flow is It may be moved in the direction along the center axis of the non-uniform electric field forming region or in the flow direction passing through the central portion. For example, if an lmm-long electrode is placed in a ⁇ 100 // m beam with a flow of 50 n 1 min, an analysis time of about 10 seconds can be obtained.
  • the diameter of the incident light to the inhomogeneous electric field forming region should be larger than the diameter that will interfere with differential interference or phase interference in the flow path so that optical observation is not hindered.
  • Incident light can be irradiated so that it does not hit the walls of the channel.
  • the incident light is preferably straight light or convergent light, and the diameter of the incident light is set or adjusted so as to be larger than the diameter that interferes with differential interference or phase interference in the flow path. obtain.
  • the sample Upon completion of the observation, the sample is discharged from the liquid outlet part of the flow path by the above suction means.
  • the obtained data is subjected to data correction based on the sample shape, position, flow, electric field distribution, electrode shape, etc.
  • the electrophoretic mobility is calculated.
  • detailed analysis can be performed on the target fine particles based on changes in initial acceleration and speed. A profile for each frequency is created and analyzed in detail.
  • R t and the step of correlating the characteristic of the calculated Yuden mobility and particulate may further comprise a.
  • the method of the invention for example, after about E to measure the position R t of the fine particles.
  • the chip of the present invention has a plurality of flow paths, the conditions are changed. Measurements can be performed sequentially or simultaneously. In addition, multiple samples can be measured with a single measurement in the same channel.
  • fine particles can be separated and reused based on the calculated data.
  • a database can be created by accumulating data obtained for fine particles. It is also possible to characterize fine particles by comparing the measured data of individual fine particles with the obtained database.
  • the chip or device of the present invention when used, it is possible to change the electric field frequency and the electric field shape by one measurement in the same flow path. Specifically, the alternating current applied to the electrode is changed over time according to the movement of the fine particles to be measured. By changing the electric field in this way, it is possible to acquire a complex spectrum and to acquire multiple frequency data by searching for the optimal migration frequency and changing the electric field according to the program.
  • Example 1 Manufacture of a Quadrupole Micro Capillary / Dielectric Migration Chip
  • a quadrupole microcapillary / dielectric electrophoresis chip having a capillary as shown in FIG. 2 was manufactured.
  • epoxy resin EP I CLON N—690_75M
  • EP I CLON B 650 the hole where the electrode and each electrode are placed was produced by molding, sealing, cutting, or laser processing.
  • an epoxy adhesive (ARALD I TE HV 9 53U, AW 106) to the placement hole processed into epoxy resin, and place a positioning jig ( ⁇ 0 ⁇ 07 corresponding to the capillary diameter) on a part of the pillar. ⁇ 0.14 (made of super steel or nylon), and then each electrode was inserted into the placement hole.
  • Heat curing was performed at 60 ° C for 60 minutes with a dryer (Hello Soyokaze made by I SUZU). After that, the electrode was wired with a conductive paste or the part extended from the electrode was fixed on the chip and wired.
  • a positioning jig (flow channel) was placed between the electrodes.
  • These electrodes and positioning jig are made of epoxy resin (EP I CLON N-690-75M, EP I CLON B 650) with the above dryer 120. After pre-curing with C for 2 hours, the positioning jig was extracted. Next, wiring was done by fixing the conductive '["raw paste or the part extended from the electrode on the chip on the chip. When the electric field strength needs to be changed in the Z-axis direction, An angle is formed on the exposed surface, or the shape of the arrangement hole or arrangement jig made in the epoxy resin is tapered In the following examples, the body mounted with the chip manufactured in this example The operation procedure for measuring the dielectrophoretic mobility was as follows.
  • KC 1 solution (conductivity 33.5 mS / m) in the tip (spindle diameter 100 / zm tip) and inject the sample into the solution reservoir at the top of the tip.
  • S-ny D FW a system observation system as a moving image using a 40x lens in the system observation system.
  • X700 or PI XERA 600 SL—Start observing sample behavior with a CU digital camera.
  • the set electrophoretic electric field is generated to start the electrophoresis.
  • Dielectrophoresis profiles were obtained for commercial yogurt and yeast.
  • the applied voltage was 5 V, and KCl solution (conductivity 33.5 mS / m) was used as the solvent.
  • the applied frequency was 1 kHz to 10 MHz. The results are shown in FIG.
  • yoghurt bacteria are forward migrating, yeast
  • the area and cylindrical shape covering the electrode by changing the opening diameter of the electrode support wall other than the portion in direct contact with the electrode Changed the position of the wall of the Capillary. Specifically, if the electrode exposed surface is changed to a fan-shaped arc of 120 degrees from the center of the electrode without changing the position of the electrode, the capillary wall diameter is ⁇ 0.15, and the 180-degree fan-shaped arc If you do ⁇ 0.20. .
  • the behavior of fine particles can be measured from the dielectrophoretic mobility.
  • the method of the present invention it is possible to analyze one individual fine particle without requiring a large amount of sample, and the analysis speed is high. Furthermore, if a database is created, it is possible to identify fine particles and to estimate (discriminate) substances.
  • the sample used for analysis is reused.
  • the fine particles that have been subjected to analysis can also be subjected to further precision analysis.
  • the chip of the present invention provided with a flow path and a non-uniform electric field generating means can be used in combination with a commercially available observation apparatus (such as an optical microscope) for simple analysis.
  • a plurality of channels are easily provided in a single chip, a plurality of samples can be measured simultaneously under the same conditions or a single sample can be measured simultaneously under different conditions. Therefore, in particular, quality control of biological microparticles (DNA of 40 kbp or more, huge proteins of 100 million Da or more such as membrane proteins, cells, bacteria, cell membranes, etc.), activity measurement, drug efficacy determination, tailor-made It is useful for applications in fields such as medicine.

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  • Engineering & Computer Science (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne une puce de diélectrophorèse à micro-réseau pour mesurer le comportement d'une particule et qui comprend un canal de flux ayant une partie d'introduction du liquide et une partie d'éjection de liquide, ainsi qu'un moyen de génération de champ électrique non homogène pour générer une région de champ électrique non homogène dans au moins une partie du canal de flux. Le champ électrique non homogène est généré symétriquement par rapport à l'axe central ou à la partie centrale du champ électrique non homogène, et un champ électrique homogène est généré dans une direction le long de l'axe central ou dans la direction du flux passant par la partie centrale ou un champ électrique dont la force est augmentée/diminuée ou différente le long de l'axe central ou le long de la direction de flux passant par la partie centrale est généré. Avec cela, une mesure simple et rapide du comportement d'une particule dans différents champs électrique est possible à un moment sous forme de mobilité diélectrophorétique.
PCT/JP2007/055169 2006-03-10 2007-03-08 Dispositif de dielectrophorese WO2007105784A1 (fr)

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EP2830740A4 (fr) * 2012-03-27 2016-03-23 Univ California Trieur cellulaire par dep, tridimensionnel, à puce complète, continu, et procédé de fabrication correspondant
CN108267483B (zh) * 2018-01-29 2023-10-27 重庆科技学院 一种测试多铁性液体性能的装置及方法
CN108267483A (zh) * 2018-01-29 2018-07-10 重庆科技学院 一种测试多铁性液体性能的装置及方法
CN108287179A (zh) * 2018-01-29 2018-07-17 重庆科技学院 一种多铁性液体的性能测试装置及方法
CN108254412A (zh) * 2018-01-29 2018-07-06 重庆科技学院 一种多铁性液体的测试装置及方法
CN108254413A (zh) * 2018-01-29 2018-07-06 重庆科技学院 一种测试多铁性液体的装置及方法
CN108254412B (zh) * 2018-01-29 2023-10-27 重庆科技学院 一种多铁性液体的测试装置及方法
CN108254413B (zh) * 2018-01-29 2023-10-27 重庆科技学院 一种测试多铁性液体的装置及方法
CN108287179B (zh) * 2018-01-29 2023-10-27 重庆科技学院 一种多铁性液体的性能测试装置及方法
CN111589588A (zh) * 2019-02-20 2020-08-28 李庆宪 插板电极式介电电泳选矿设备
CN111589588B (zh) * 2019-02-20 2023-09-26 李庆宪 插板电极式介电电泳选矿设备

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