JP2000105218A - Electromagnetrophoretic analytisis method and device for micro particulate in liquid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To conduct detail separation/characterization for a microbody such as a molecule, and to easily carry out fractionation utilizing migration, by applying an electric field to the microbody dispersed in a dispersion medium and applying a magnetic field orthogonally to the electric field to conduct electrophoresis for the microbody in the magnetic field. SOLUTION: An Ag/AgCl electrode, for example, is provided by a cell holder in both end parts of a cell, a current flows to the i-direction in the figure, and a magnet is provided to apply a magnetic field H in a direction perpendicular to it. A light source is provided in a lower side of the cell to observe migration thereby from an upper side of the cell. Any of a charged microbody, a microbody charged by applying the electric field or the magnetic field, or an uncharged body is acceptable for the microbody of a migration object. A particulate of a polymer material such as polystylene, an inorganic particulate such as carbon, a molecule or a molecule flocculate, and a protein such as an enzyme are mentioned as an example.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for applying an electric field to a minute object dispersed in a dispersion medium and applying a magnetic field in a direction perpendicular to the electric field to cause the minute object to electrophoretically move in the magnetic field.
The present invention relates to an apparatus therefor, a method for separating a minute object by the method, and a separation apparatus therefor.

[0002]

2. Description of the Related Art Electrophoresis of charged or charged minute objects has been widely applied as an analysis means or a separation means. These electrophoresis methods have been performed by applying an electric field to a dispersion medium containing minute objects, but did not apply a magnetic field simultaneously with the electric field.

[0003] Although a theoretical formula for the electrophoretic force applied to a submerged particle in a uniform electric field by a uniform magnetic field has been previously proposed (A. Kolin, Science, 1953, 117, 134.), it is not possible to consider the actual migration. Almost never done. Then, the present inventors examined the influence of a magnetic field on the electrophoretic behavior of a minute object in a dispersion medium, and examined the possibility as a new separation and characterization method of a minute object in a liquid.

[0004]

SUMMARY OF THE INVENTION The present invention examines the effect of a magnetic field on the electrophoretic behavior of a minute object such as a molecule or a fine particle in a dispersion medium, and examines the effect of a magnetic field in a dispersion medium, particularly a solution-type dispersion medium. An object of the present invention is to provide a new electrophoresis method and separation method for an object. More specifically, the present invention relates to a method for causing a minute object dispersed in a dispersion medium to electrophores the minute object in an electromagnetic field by applying an electric field and a magnetic field in a direction perpendicular to the electric field, an apparatus therefor, and a method therefor. To provide a method and apparatus for separating a minute object.

[0005]

SUMMARY OF THE INVENTION The present invention relates to a method for applying an electric field to a minute object dispersed in a dispersion medium and applying a magnetic field in a direction perpendicular to the electric field to cause the minute object to undergo electrophoresis in the magnetic field. . Further, the present invention relates to an apparatus for applying an electric field to a minute object dispersed in a dispersion medium and applying a magnetic field in a direction perpendicular to the electric field to cause the minute object to electrophoretically move in the magnetic field. Furthermore, the present invention provides a method for separating a minute object by a method in which an electric field is applied to a plurality of kinds of minute objects dispersed in a dispersion medium, and a magnetic field is applied in a direction perpendicular to the electric field and the minute object is subjected to electrophoresis in the magnetic field. A method and an apparatus therefor.

Among the magnetic permeability μ, the current density j, and the magnetic field strength H, the force F received by an object having a certain volume element V by a magnetic field and an electric field perpendicular thereto is given by the following equations (1) and (2). ). dF = (μHj) dV (1) F = (μHj) V (2) When the current density j is different between the medium and the minute object in the medium, the forces applied to the medium will be different. Due to the difference, the minute object migrates in the medium. The relationship between the minute object in the medium and the current density of the medium is expressed by the following equation (3) from Maxwell's equation. j ₁ = j ₂ (3σ ₁ / (2σ ₂ + σ ₁ )) (3)

Here, j ₁ is the current density in the minute object, j
₂ is the current density in the medium, σ ₁ is the electrical conductivity of the minute object,
σ ₂ indicates the electric conductivity of the medium. According to the above equation (2), the force F ₁ received by the minute object having the volume V in the medium in the electromagnetic field and the force F ₂ received by the medium are expressed by the following equations (4) and (5), respectively. Will be. F ₁ = μ ₁ Hj ₂ V (3σ ₁ / (2σ ₂ + σ ₁ )) (4) F ₂ = μ ₂ Hj ₂ V (5) where μ ₁ is the magnetic permeability of the minute object, and μ ₂ is This is the magnetic permeability of the medium. Therefore, the relative force F (that is, F ₁ −F ₂ ) received by the minute object in the medium in the electromagnetic field is expressed by the following equation (6). F = F ₁ −F ₂ = Hj ₂ V (μ ₁ (3σ ₁ / (2σ ₂ + σ ₁ )) − μ ₂ ) (6) Here, the magnetic permeability of the medium and that of the minute object are equal or almost equal.
That is, assuming that μ ₁ ≒ μ ₂ = μ and n = σ ₁ / σ ₂ , the relative force F is expressed by the following equation (7). F = −2 μHj ₂ V ((1-n) / (2 + n)) (7)

If the force F is compared to a gravitational field,
Water material is such as under gravity F ₁ and the buoyancy F _2. However, the force F which has been described above, since a force received by the electromagnetic field, in the following, the F ₁ is referred to as electromagnetic force, will be an F ₂ is referred to as electromagnetic buoyancy. FIG. 1 shows the relationship between the above-described forces received by a minute object in an electromagnetic field.

Further, as a minute object, its electric conductivity σ ₁
Is 0, the electromagnetic force F
₁ is 0, the result, only the electromagnetic buoyancy F ₂ acts substantially in very small objects. That is, the force F in this case is expressed by the following equation (8). F = −μ ₂ Hj ₂ V (8) Therefore, when a minute object having substantially zero electrical conductivity is dispersed in a medium having a magnetic permeability of μ ₂ and placed in an electromagnetic field, the minute object the volume V, and the force F proportional to the current density j ₂ in the medium, it takes a very small objects.
Here, if the current density is kept constant, the applied force will differ according to the volume of the minute object, and electrophoresis according to the volume will be observed. Theoretically, σ ₁ = 0 (n
= 0), but σ ₁ is not 0 even in the case of polystyrene, and electrophoresis also depends on σ ₁ , that is, the value of n.

The migration speed v at this time is given by the following equation (9).
F = 6πηRv (9) (where η is the viscosity of the medium and R is the radius of the minute object) and the above equation (7), and the following equation (7) 10). v = (2/45) · (μHj ₂ R ² / η) ((1-n) / (2 + n)) (10) (where, μ ₂ is the magnetic permeability of the medium, and H is the strength of the magnetic field. , J ₂ is the current density in the medium, η is the viscosity of the medium, and R is the radius of the minute object.)

The above situation is a case of a uniform magnetic field. When the magnetic field is not uniform, a force corresponding to the magnetic field gradient acts on the medium and the minute object. The force (magnetic field attraction force) Fm acting on the minute object having the volume V is expressed by the following equation (11). Fm = Vμ ₀ χ (H · (dH / dx)) (11) where μ ₀ is the magnetic permeability of vacuum, μ ₀ = 1 in the CGS unit system, χ is the magnetic susceptibility, and (dH / Dx) indicates the magnetic field gradient.

This force Fm is not so problematic in a substance having a small magnetic susceptibility χ, as can be seen from the equation (11), but is particularly in the case of a paramagnetic substance having a large magnetic susceptibility よ う such as an aqueous manganese chloride solution. Will work as a great force. When the minute object in the medium in the inhomogeneous magnetic field is a non-magnetic substance, there is no force acting on the minute object as can be seen from the above equation (11), so the apparent force Fm acting on the medium having the same volume is It will be applied to the minute object in the opposite direction. This is based on the same concept as the electromagnetic buoyancy described above. Therefore, a force F applied to a minute object made of a non-magnetic substance in a medium made of a magnetic substance in a non-uniform magnetic field is expressed by the following equation (12). F = −Vμ ₀ χ (H · (dH / dx)) (12) where μ ₀ is the magnetic permeability of vacuum, χ is the magnetic susceptibility of the medium, and (dH / dx) is the magnetic field gradient. .

The present inventors have used polystyrene particles and carbon sphere particles as minute objects, and studied the effect of a magnetic field on the electrophoretic behavior of these minute objects. FIG. 2 shows the cells used in the experiment. The unit in FIG. 2 is mm. The magnet used in this experiment was a neodymium iron boron magnet having a length of 16 mm, a width of 5 mm, a height of 5 mm and a surface magnetic field of about 0.3 T, thereby generating a uniform magnetic field.

The electrophoresis experiment apparatus used in this experiment is shown in FIG. 3, FIG. 4, and FIG. FIG. 3 shows a cell holder in which electrodes (for example, Ag / AgCl electrodes and the like) can be installed in both openings of the cell shown in FIG. FIG. 4 shows the relationship between the cell holder and the magnet. That is, electrodes are provided by cell holders at both ends of the cell, current flows in the direction indicated by i in FIG. 4, and magnets are installed on both sides so that a magnetic field H is applied in a direction perpendicular to this. The distance between the magnets is 23
mm. A light source was provided below the cell, and the electrophoresis was observed from above the cell by the light source.
Light was applied from below by a light source built into the microscope as shown in FIG.

FIG. 5 shows an overall view of the apparatus. This device has a constant current power supply for supplying current to the cell, a microscope for observation, and a CCD for recording observation results.
A camera and a video device and a digital gauge for determining the height of the stage are provided. With this digital gauge, the height of the stage can be adjusted on the order of μm. The position of the focused stage can be measured on the order of μm using this digital gauge, but when a solution is placed in the cell, it is not possible to use the value of the digital gauge as it is in the cell due to the difference in the refractive index. could not. For example, the difference in stage height when focusing on the top and bottom surfaces of the cell is about 190 μm
Which is smaller than the actual cell depth of 250 μm (see FIG. 2). For this purpose, it is necessary to correct the value measured by the digital gauge, and the following equations (13) and (1)
The correction was made by the equation shown in 4).

To determine the depth of the particles in the cell, first focus on the upper and lower surfaces of the cell and record the height of the stage. Next, focus on the particles and record the stage height. Then, the ratio (Dr) of the relative depth of the particles was determined by the following equation (13), and the height (h _D ) from the lower surface of the cell was determined from the value of Dr by the following equation (14). Dr = (h ₁ −h ₃ ) / (h ₂ −h ₃ ) (13) (where h ₁ is the height of the stage when the particle is focused, and h ₂ is the focus on the upper surface of the cell. the height of the stage when combined, _{h 3} is the height of the stage when focused on the lower surface of the _{cell.) h D = Dr × 250μm} (14)

Three kinds of polystyrene particles having a particle size of 3 μm, 15 μm and 20 μm were electrophoresed as minute objects. As a medium, a 1M KCl solution was used. FIG. 6 shows the result. The vertical axis in FIG. 6 indicates the migration speed v (μm /
Second), and the horizontal axis represents the current i (μA).

As can be seen from FIG. 6, the migration speed v is proportional to the current i, which is a result as shown by the above equation (9). In this experiment, the flow of the medium was not observed, and the migration of only a minute object was observed. The migration speed v increases as the particle diameter increases, which indicates that the electromagnetic buoyancy is in good agreement with the dependence on the particle volume. The inclination of a minute object having a particle diameter of 20 μm is approximately 1.74 times the inclination of a minute object having a particle diameter of 15 μm, which is a square magnification of the radius (1.78).
Times). In addition, particle size 3
Electrophoresis of a micrometer micro object could not be observed.

FIG. 7 shows the result of calculation of the migration speed v in this experiment assuming that n = 0 in the equation (9). When the magnetic field strength H is 1050 Oersted (Oe),
The medium has a viscosity η of 8.788 × 10 ⁻³ poise (pois).
e), calculated in CGS unit system. Although the calculated value in FIG. 7 and the measured value in FIG. 6 show a larger difference as the current value increases, the tendency is consistent, and quantitatively, the measured value is n = It can be seen that the calculated value matches the experimental value when 0.323 is set.

Next, an experiment of electrophoresis in a non-uniform magnetic field was performed. In this experiment, two kinds of minute objects having different electric conductivity were used. Polystyrene latex (carboxylate) particles with low conductivity (hereinafter abbreviated as polystyrene particles)
And carbon sphere particles, which are conductive particles, were used as minute objects. Each preparation method is shown below. First, polystyrene particles (with a radius of 1.5 μm) are obtained by converting a commercially available latex sample into purified water (water obtained by purifying distilled water with MILLIPORE) (hereinafter abbreviated as water), or the number of particles that can be easily observed with an aqueous KCl solution. The diluted one was used (particle number 3.3
6 × 10 ⁵ cm ⁻³ ). Actually, 20 μl of a latex sample was added to 100 cm ³ of water. Samples were usually stored refrigerated and returned to room temperature (25 ° C.) before measurement.

Next, using carbon column beads of Biotech Research as carbon sphere particles, about 0.05% (W /
W). However, since water alone floated and did not disperse, the neutral surfactant Triton X-100 (polyoxyethylene (10) octyl phenyl ether (CH ₃ (CH ₂ ) ₇ C ₆ H) was used.
₄ (OCH ₂ CH ₂ ) ₁₀ OH (a neutral surfactant composed of ₁₀ OH) is added to a concentration of about 0.01% (V / V), and shaken for about 1 hour with a shaker to obtain a good dispersion. Obtained. Although the radii of the particles varied, only fairly small particles (radius about 1 μm) were dispersed for some time.
Actually, about 100 cm ³ of water,
5 g and 10 μl of Triton X-100 were added.

FIG. 8 shows an experimental apparatus used in this experiment. The experimental apparatus in FIG. 8 is almost the same as the experimental apparatus in the uniform magnetic field shown in FIG. 5 except for the cell part and the digital gauge part. Here, for the sake of convenience, the coordinate axes were placed on the cell (inner diameter 73 × 23 × 1 mm) shown in the upper right of FIG.
The length direction was the X axis, the width direction was the Y axis, and the depth direction was the Z axis. The cell was fixed to an aluminum frame, and the aluminum frame was fixed horizontally on the stage so that it could be moved in parallel to the XY directions. The state of migration of the particles was observed from directly above the cell using an optical microscope. The magnification is 300 times (eyepiece lens × 15, objective lens × 20). Since this microscope has a graduated fine screw for focus adjustment, the depth was determined from the value.

The procedure for measuring the electrophoretic behavior is basically the same as the procedure for measuring the electrophoretic behavior described above. What is unusual is that a magnet (16 × 16 × 5 mm) is installed under the cell. Since this magnet is always directly below the objective lens irrespective of the movement of the stage, there is a uniform magnetic field perpendicular to the horizontal uniform electric field in the field of view of the microscope (see FIG. 9). A very strong neodymium iron boron magnet was used for the magnet (with a magnetic flux density of about 0.36
T). FIG. 9 shows the cell part of this experiment.

First, the pH and conductivity of the sample are measured before measurement. After the cell is filled with the sample, it is fixed on a stage, and a groove that serves as a depth reference is first searched with a microscope. Next, the XY stage is used to search for the focused particle by moving the XY stage from the upper groove to a steady level using a fine movement screw. If particles are found, border the video and apply voltage (approximately 5
0V). When the electrophoresis behavior is green, stop applying the voltage and stop the video. In this way, several particles are measured, the polarity of the electrode is changed, and the upper and lower steady levels are measured, respectively, and one sample is measured in these four processes.
The conductivity of the measured sample is measured once again.

Since it was felt that the flow of the sample flowing through the cell was somewhat delayed by adding Triton X-100, the viscosity of the aqueous solution of Triton X-100 was examined. For the measurement, a relative viscosity with respect to water at 25 ° C. was obtained by using a Jubelod viscometer.

First, the state of electrophoresis using only electricity without applying a magnetic field was observed. The result is shown in FIG. The photo shows the positive electrode on the left and the negative electrode on the right.
Is an image of the polystyrene latex particles using the image, the right side is when a voltage is applied (0 second), the middle is after 8 seconds, and the left side is an image after 16 seconds. In this electrophoresis, the polystyrene latex particles migrated only in the X-axis direction (horizontal direction in the figure), and did not migrate in the Y-axis direction (vertical direction in the figure).

Next, when a magnetic field was applied, the migration behavior of the particles clearly changed. The results are shown in FIG. Each photograph is the same as in FIG. 10 described above, but the upper part of FIG.
Cl was not added (0 mM), the second stage was 0.1 mM KCl, and the third stage (bottom) was K
This is for Cl1 mM. Unlike the case of electrophoresis, the particles observed larger migration in the Y-axis direction (vertical direction in the figure) as the KCl concentration increased.

As shown in this photograph (FIG. 11), the particles are oblique.
Run electrophoresis. Therefore, the migration speed of the particles is set in the X-axis direction and Y-axis direction.
The difference with electrophoresis
investigated. The velocity component Vx in the X-axis direction is the same as in electrophoresis
Orientation, so the electrophoretic mobility and the electrophoretic mobility obtained from Vx
To determine whether there is a contribution from the magnetic field.
Was. In this study, the electrophoretic mobility was used as the electrophoretic velocity.
This is because a simple comparison cannot be made. Divide Vx by electric field
Migration mobility normalized to KCl concentration
Mobility is almost the same as electrophoretic mobility
did. FIG. 12 shows the result. The black circle in FIG. 12 is carbon
The white circles indicate polystyrene latex particles.
Shows the child case. The horizontal axis of FIG. 12 shows the KCl concentration (10^-3
Mol / dm³ ) Is shown as a logarithm, and the vertical axis represents the velocity component V.
x migration mobility (10^-8m²/ Sec / V)
You. FIG. 13 similarly shows the migration mobilities. From the figure
As can be seen, there is almost no difference in the migration mobility
Was. From this result, Vx in electrophoresis in the presence of a magnetic field is
It was found that it was only the contribution of electrophoresis.

The velocity component Vy in the Y-axis direction was 0 during electrophoresis. Moreover, since Vx was only electrophoresis, this Vy component is considered to be electrophoresis itself. Since the electrophoretic force is proportional to the current density, FIG. 14 shows the result of plotting Vy against the current. FIG.
Black circles indicate the case of carbon particles, and white circles indicate the case of polystyrene latex particles. The horizontal axis in FIG. 14 shows the current (10 ⁻⁶ A), and the vertical axis shows the migration speed (10 ⁻⁶ A).
^-6 m / sec). As a result, polystyrene,
Both carbon particles could be approximated to the same straight line passing through the origin. This indicates that no difference was observed in the electrophoretic force despite the two particles having completely different electrical conductivities.

The above results indicate that in the electrophoresis in a non-uniform magnetic field, the electromagnetic flow is dominant, and in a state close to an open system, the electromagnetic flow and the electrophoresis act in directions perpendicular to each other, and the Separation can be performed,
This indicates that the closed system can be used as a power source for bulk flow. Also, in electrophoresis in a uniform magnetic field, no flow occurs, and electrophoretic separation of particles occurs due to electromagnetic buoyancy, and the migration speed is proportional to the square of the particle radius, proportional to the current, and the difference between this and electrophoresis. The combination is also possible, indicating that electrophoretic analysis based on particle size, charge, and conductivity is expected. Therefore, the present invention, by a combination with a dispersion medium,
The present invention shows a possibility as a separation or characterization method of minute objects in an electromagnetic field, and the present invention provides a new separation and characterization method different from the conventional electrophoresis method using only an electric field. Things.

The minute object to be electrophoresed in the present invention may be a charged minute object, a minute object charged by applying an electric or magnetic field, or a non-charged object. It may be something. It is preferable that the minute object has a small amount of conductivity. Examples of the minute object in the present invention include, for example, particles of a polymer substance such as polystyrene, particles of an inorganic substance such as carbon,
Examples include molecules and aggregates of molecules, proteins such as enzymes, peptides, and microorganisms such as bacteria and cells. These minute objects may be provided with a marker such as a fluorescent substance or a radioactive substance as necessary. These minute objects can be separated by the method of the present invention not only by conventional separation by electrophoresis but also by their size, for example, volume and shape.

The medium is not particularly limited as long as it has electrical conductivity. The medium may be liquid or gel, but an aqueous solution is preferred. Further, other additives such as a surfactant can be added to the dispersion medium as needed.

The method of the present invention is characterized in that a magnetic field is applied in a direction perpendicular to the electric field, but the direction in which the magnetic field is applied can be appropriately changed according to the medium and the minute object to be separated. The magnetic field can be applied not only from one direction but also from multiple directions as needed. The magnetic field may be uniform throughout the cell, but may be non-uniform.

The cell used in the present invention is preferably a closed cell, but need not be a closed cell, and can be appropriately designed according to an electric field and a magnetic field. In addition, the cell may have a fractionation unit capable of fractionating a minute object according to two-dimensional migration. Conventional electrophoresis is a one-dimensional electrophoresis, and it is difficult to fractionate. However, since the electrophoresis of the present invention is a two-dimensional electrophoresis, a fractionation part, for example, a partition for the fractionation, is used. By providing a separation unit provided with a plate or the like, it becomes possible to directly separate the separated minute object into the separation unit.

[0035]

EXAMPLES Next, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Apparatus A medium and a small object are put in a cell shown in FIG. 2 having a length of 0.25 mm and a width of 6 mm and a length of 6 mm, and electrodes are provided at both ends of the cell shown in FIG. Can be fixed to the cell holder. As an electrode, a silver wire having a diameter of 1 mm was placed in a Teflon tube, and electrolysis was performed using this as an anode and a platinum wire as a cathode. When a constant current of 100 μA is passed for 5 to 10 minutes,
Silver chloride adhered to the tip of the silver wire. This was used as a reversible electrode. When fixing the cell containing the medium and the minute object to the cell holder, the solution in the cell is sealed, and when the solution is sealed in the cell, work is performed in the solution to prevent air bubbles from entering, and the screw is screwed. Liquid leakage was eliminated by tightening with. The cell holder to which the cell was fixed was set between the magnets shown in FIG. 4 so that a magnetic field was applied to the electric field from the vertical direction. A neodymium iron boron magnet having a length of 16 mm, a width of 5 mm, and a surface magnetic field of about 0.3 T was used as the magnet. The cell holder set in the magnetic field was placed on the microscope shown in FIG. 5, and a digital gauge was set. FIG. 5 shows an overall image of the apparatus used in the experiment. Electrodes were connected to both ends of the cell, the cell was sandwiched between two magnets, and light was applied from below by a light source built into the microscope.

The following was used for the apparatus shown in FIG. Constant current power supply Fuso Seisakusho HECS 317B Voltmeter Takeda Riken Digital multimeter TR6843 Microscope Nikon Biological microscope LABOPHOT stage Central precision machine Small twin axis controller MMC-2 Central precision machine Small automatic pulse stage MM stage CCD camera SONY CCD-IRIS Digital gauge PEACOK No. 107 Light source OLYMPUS MODELGPS Gauss meter Yokokawa Electric Type3251

(2) Reagent and Measurement Using the apparatus described in the above (1), the particle size was 3 μm,
μm polystyrene latex particles (diameter 14.571μ)
m, standard deviation 1.657 μm) (hereinafter abbreviated as 15 μ polystyrene particles) and 20 μm polystyrene latex particles (diameter 22.001 μm, standard deviation 2.712 μm).
m) (hereinafter, abbreviated as 20 μ polystyrene particles) were electrophoresed as minute objects. As a medium, a 1M KCl solution was used. This was diluted to an easily observable particle number. The samples were stored refrigerated at Shindan and returned to room temperature (25 ° C.) before measurement. FIG. 6 shows the result. The vertical axis in FIG. 6 shows the migration speed v (μm / sec), and the horizontal axis shows the current i (μA).

Example 2 (1) Apparatus An experiment of electrophoresis in a non-uniform magnetic field was performed. The apparatus for this experiment is shown in FIG. In this experiment, constant voltage power supply, optical microscope, CCD camera, Briggs cell (Briggs cell)
-Cell) An apparatus for measuring ζ potential (Mitamura Riken Kogyo) consisting of one set was used. Here, for convenience, a coordinate axis is placed on the cell (inner diameter 73 × 23 × 1 mm) shown in the upper right of FIG.
The length direction was the X axis, the width direction was the Y axis, and the depth direction was the Z axis. The cell was fixed to an aluminum frame, and the aluminum frame was fixed horizontally on the stage so that it could be moved in parallel to the XY directions. The state of migration of the particles was observed from directly above the cell using an optical microscope. The magnification was 300 times (eyepiece lens × 15, objective lens × 20). Since the microscope was provided with a graduated fine screw for focus adjustment, the depth was determined from the value.

The apparatus used in this experiment is shown below. pH meter (glass electrode type hydrogen ion concentration meter) HORIBA, Ltd. Model F-14 F-70 Conductivity meter (CONDUCTIVITY METER) TOA ELEOTROCHEMICAL MESURING INSTRUMENTS CM-40V Conductivity cell CT-54101B Gauss meter Yokokawa Electric Type 3251.

(2) Reagent In order to see the difference in electrophoretic force, two kinds of particles having different electric conductivity were used in this experiment. These are polystyrene latex (carboxylate) particles (hereinafter abbreviated as polystyrene particles), which are insulators, and carbon sphere particles, which are conductor particles. Each preparation method is shown below. First, polystyrene particles (with a radius of 1.5 μm) are obtained by diluting a commercially available latex sample with purified water (hereinafter abbreviated as water) obtained by purifying distilled water with MILLIPORE or a KCl aqueous solution to a particle number that is easily observed. (The number of particles
3.36 × 10 ⁵ pieces · cm ⁻³ ). Actually 100cm
20 μl of the latex sample was added to the water of No. ³ . The samples were stored refrigerated at Shindan and returned to room temperature (25 ° C.) before measurement.

Next, using carbon column beads of Biotech Research as carbon sphere particles, about 0.05% (W /
W). However, since water alone floated and did not disperse, Triton X-100, a neutral surfactant, was added to a concentration of about 0.01% (V / V), and was shaken for about 1 hour. Upon shaking, a good dispersion was obtained. Although the radii of the particles varied, only fairly small particles (radius, about 1 μm) were dispersed for some time. In fact, for 100 cm ³ of water,. About 0.05 g of carbon spheres and 10 μl of Triton X-100 were added.

(3) Method for Measuring Electrophoretic Behavior First, before measurement, the pH and conductivity of the sample are measured. Then, the sample is thoroughly stirred before being put into the cell, and the cell is filled with the sample so that no air remains inside the cell. Since the particles have a higher density than water and gradually sink, the measurement is carried out finely thereafter. After the cell is filled with the sample, it is fixed on a stage, and a groove that serves as a depth reference is first searched with a microscope. Next, the XY stage is used to search for the focused particle by moving the XY stage from the upper groove to a steady level using a fine movement screw. Once the particles are found, record the video and apply voltage (about 50V). When the electrophoresis behavior is green, stop applying the voltage and stop the video. In this way, several particles are measured, the polarity of the electrode is changed, and the upper and lower steady levels are measured, respectively, and one sample is measured in these four processes. The conductivity of the measured sample is measured once again.

(4) Method for Measuring Electrophoretic Behavior The operation is basically the same as the method for measuring electrophoretic behavior in (3). What changes is that a magnet (16 × 16 ×
5 mm). Since this magnet is always directly below the objective lens irrespective of the movement of the stage, there is a non-uniform magnetic field perpendicular to the horizontal uniform electric field in the field of view of the microscope (FIG. 9). A very strong neodymium iron boron magnet was used as the magnet (the magnetic flux density was about 0.36 T above the glass of 1 mm).

(5) Measurement of Viscosity Since the flow of the sample flowing through the cell seemed to be slightly delayed due to the addition of Triton X-100, the viscosity of the aqueous solution of Triton X-100 was examined. For the measurement, a relative viscosity with respect to water at 25 ° C. was obtained by using a Jubelod viscometer.

(6) Measurement Results First, when a magnetic field was applied, the migration behavior of the particles clearly changed. The particles migrated in a direction different from the direction of electrophoresis. The appearance is shown in a series of photographs (FIGS. 10 and 11).
In each of the photographs, the left side is the positive electrode and the right side is the negative electrode, which is an image of polystyrene latex particles. In electrophoresis, electrophoresis is performed only in the X-axis direction as shown in the photo of FIG. 10, but in electrophoresis, electrophoresis is also performed in the Y-axis direction as in the photo of FIG.
The migration speed in the Y-axis direction increased as the concentration increased. In the electrophoresis, as shown in the photograph of FIG. 11, particles migrate diagonally. Therefore, the electrophoretic velocity of the particles was vector-decomposed into the velocity in the X-axis direction and the velocity in the Y-axis direction, and the difference from electrophoresis was examined.

KCl concentration dependence of the migration mobility of the velocity component Vx parallel to the electric field The velocity component Vx in the X-axis direction is in the same direction as the electrophoresis.
By comparing the electrophoretic mobility with the electrophoretic mobility determined from Vx, it was examined whether or not there was any contribution from the magnetic field. The electrophoretic mobility was used because a simple comparison was not possible with the electrophoretic velocity. The results are shown in FIGS. As can be seen from the figure, there was almost no difference in the migration mobility. From this result, it was found that Vx in electrophoresis in the presence of a magnetic field was only an electrophoretic contribution. This is consistent with the fact that the electrophoretic value acts in a direction perpendicular to the electric and magnetic fields.

Current dependency of velocity component Vy perpendicular to the magnetic field The velocity component Vy in the Y-axis direction was 0 during electrophoresis. Moreover, since Vx was only electrophoresis, this Vy
Is considered to be the electrophoresis itself. Since the electrophoretic force is proportional to the current density, this Vy was plotted against the current (FIG. 14). As a result, both polystyrene and carbon particles could be approximated to the same straight line passing through the origin. This indicates that no difference was observed in the electrophoretic force despite the two particles having completely different electrical conductivities.

[0049]

According to the electrophoresis method of the present invention, since a minute object moves not only in the direction of an electric field but also in the direction of a magnetic field perpendicular to the electric field, more detailed separation and characterization of minute objects such as molecules can be performed. It can be carried out. In addition, since electrophoresis is two-dimensional, fractionation by electrophoresis can be easily performed.

[Brief description of the drawings]

FIG. 1 schematically shows the relationship between the electromagnetic force and electromagnetic buoyancy of a minute object in a uniform magnetic field.

FIG. 2 shows a cell used in the first embodiment of the present invention.

FIG. 3 shows a cell holder used in Example 1 of the present invention.

FIG. 4 shows the cell holder used in Example 1 of the present invention placed in a magnetic field.

FIG. 5 shows the apparatus of the present invention used in Example 1 of the present invention.

FIG. 6 is a graph showing measured values of the relationship between the migration speed of a minute object and the current in a uniform magnetic field.

FIG. 7 is a graph showing calculated values of a relationship between a migration speed of a minute object and a current in a uniform magnetic field.

FIG. 8 shows the apparatus of the present invention used in Example 2 of the present invention.

FIG. 9 shows a state where the cell holder used in Example 2 of the present invention is placed in a magnetic field.

FIG. 10 is a photograph instead of a drawing, showing a state of electrophoresis in Example 2 of the present invention.

FIG. 11 is a photograph, instead of a drawing, showing the state of electrophoresis in Example 2 of the present invention. The top row shows the case where the KCl concentration is 0 mM.
In the case of 1 mM, the bottom row shows the case where the KCl concentration is 1 mM.

FIG. 12 is a graph showing the electrophoretic mobility and KCl of the velocity component Vx parallel to the electric field in the electrophoresis of Example 2 of the present invention.
It is a graph of the relationship between the concentrations based on the actually measured values. White circles indicate polystyrene particles, and black circles indicate carbon particles.

FIG. 13 is a graph showing the relationship between the electrophoretic mobility and the KCl concentration in the electrophoresis of Example 2 of the present invention based on the actually measured values. White circles indicate polystyrene particles, and black circles indicate carbon particles.

FIG. 14 is a graph showing the relationship between the migration speed of the velocity component Vy perpendicular to the electric field and the current in the electrophoresis according to the second embodiment of the present invention based on the actually measured values. White circles indicate polystyrene particles, and black circles indicate carbon particles.

Claims (8)

[Claims] 1. A method in which an electric field is applied to fine particles dispersed in a dispersion medium and a magnetic field is applied in a direction perpendicular to the electric field to cause a minute object to undergo electrophoresis. 2. The method according to claim 1, wherein the magnetic field is an inhomogeneous magnetic field. 3. The method according to claim 1, wherein the minute object is a conductive fine particle. 4. The method according to claim 1, wherein the dispersion medium is an electrolyte solution. 5. Apparatus for performing the method according to claim 1. 6. A method for separating a minute object by the method for electrophoresing a minute object according to claim 1. 7. A separation device for performing the method according to claim 6. 8. The separation apparatus according to claim 7, wherein a fraction for separating a minute object is provided at a position perpendicular to the electric field.