JP3513591B2 - Method and apparatus for separating magnetic traps in a liquid under a magnetic gradient - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention utilizes a magnetic gradient generated in a minute region by a small permanent magnet or an external magnetic field to magnetically trap magnetic particles such as biological particles suspended in a solvent such as an aqueous paramagnetic metal ion solution. The present invention relates to a method and an apparatus for separating magnetic particles in a liquid under a magnetic gradient, in which particles are separated according to particle size and magnetic susceptibility by controlling the force and the flow rate of a particle suspension solvent.

[0002]

2. Description of the Related Art Separation methods that use a magnetic field as an external field are
Since the 970's, rotating disk type magnetic separation and high gradient magnetic separation (HGMS) have been put to practical use. In those methods, mixed materials are separated using various magnetic gradients.
Particularly, in HGMS, an external magnetic field is vertically applied to a ferromagnetic wire having a wire diameter of about 10 to 100 μm to generate a very large magnetic gradient around the magnetic wire, so that the particles to be separated have a smaller magnetic weakness. Expanded to things. Also,
With the recent development of superconducting magnets, larger magnetic field strength and magnetic field gradient can be used, and magnetic separation is expected as an analytical method for environmental chemistry and biochemistry.

[0003]

SOLUTION: The conventional magnetic separation methods have been focused on a large amount of substances having greatly different magnetic properties, and since it has been important to roughly separate them, it is based on the difference in the size and magnetic susceptibility of each particle. Precise separation was not considered. However, for example, in blood cell separation in blood, there is a demand for accuracy with which one out of 1,000 can be separated. The present invention has been completed with the aim of achieving highly accurate separation based on the individual properties of particles.

[0004]

A method of separating magnetic particles in a magnetic gradient in a magnetic gradient according to the present invention is such that fine particles of a plurality of sizes are suspended in a solvent, and the fine particle suspension solvent is caused to flow in a predetermined magnetic field region in a predetermined direction. A magnetic trapping force acting on each fine particle is applied to the flowing fine particle suspension solvent by applying a magnetic trapping force acting in the direction opposite to the flow of the fine particle suspension solvent due to the magnetic gradient formed in the minute region, to the fine particle suspension solvent. It is characterized in that the fine particles are separated based on the size and magnetic susceptibility of the fine particles by controlling the flow velocity of.

The apparatus for separating particles flowing in a magnetic gradient according to the present invention is arranged in a pair of magnets for forming a magnetic field in a minute area and the minute magnetic field area formed between the pair of magnets, and has a plurality of sizes. A cell in which a solvent in which magnetic fine particles are suspended flows in one direction, and a trapping force that controls the magnetic trapping force that acts on each fine particle when a magnetic gradient formed in a minute region is applied to a flowing fine particle suspension solvent. The control means and a flow rate control means for controlling the flow rate of the fine particle suspension solvent are provided. The magnetic trapping force acting on each fine particle and the flow rate of the fine particle suspension solvent are controlled so that the fine particles are based on the size and magnetic susceptibility of the fine particles. Is separated.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below. (1) First, the experiment, technical analysis, and theoretical configuration that have led to the present invention will be described below. When a small permanent magnet or a pair of minute magnetic pole pieces magnetized in a magnetic field are arranged with a gap of about 400 micrometers, the magnetic field gradient generated near the end of the magnet has a square of about 100 micrometers x 100 micrometers. A glass capillary having a cross-sectional area of 2 is installed, and a water solvent or a non-aqueous solvent containing fine particles and having a constant magnetic susceptibility is flown at a constant flow rate using a microsyringe or a micropump. See FIG. 1 (a) and FIG. 1 (b). A large magnetic field gradient is generated in the vicinity of the ends of the pair of magnets or magnetic pole pieces, and the direction of the magnetic force lines is reversed at the magnetic pole ends (magnetic force density B
= 0) exists.

Since no force acts at the point where the magnetic flux density B = 0, the fine particles are trapped most stably at this point. When there is a flow in the solution, a trapping force due to the magnetic buoyancy and a viscous force due to the flow work, and due to the size of the two, for example, when the flow velocity increases, small particles flow out in order.
By observing the vicinity of the maximum trap point of magnetic flux density B = 0 with a microscope and obtaining the trap rate from the number of particles entering the area and the number of particles flowing out at a certain flow velocity, the outflow curve expected from the relational expression is obtained. To be

The above phenomenon will be described in more detail. If the solvent is weakly diamagnetic and the particles contain a ferromagnetic material, the particles will have B = 0 about 100 micrometers outside the end of the magnet.
From this point, a magnetic force acting from inside the magnet into the air gap of the magnet acts, and a viscous force that works against this acts. When the solvent is paramagnetic and the particles are weakly diamagnetic, the magnetic buoyancy acts in the direction from the void of the magnet to the point B = 0. See FIG. 1 (c).

Considering the axial direction of the capillary in FIG. 1A (hereinafter referred to as the x direction and the flow direction as the positive direction), the force that the magnetic particles receive in the non-uniform magnetic field, that is, the magnetic force is expressed as follows. To be done.

[Equation 1]

The graph of FIG. 1 (c) shows the value of B (dB / dx) obtained from the simulation. Further, since this force also acts on the medium, the particles receive the following magnetic buoyancy from the medium.

[Equation 2]

Further, when the magnetic particle suspension is caused to flow at a predetermined velocity (particle velocity v _x ), the following viscous force is applied from the solvent.

[Equation 3]

Therefore, the fine particles receive the above three forces. Substituting these forces into the equation of motion,

[Equation 4] Becomes Since the acceleration term is so small that it can be ignored, the flow velocity of the magnetic particle suspension solvent when these magnetic forces F are balanced and the magnetic fine particles stop in the solvent is expressed as follows.

[Equation 5] That is, the factor other than the magnetic field that determines the flow velocity of the magnetic particle suspension solvent when the magnetic fine particles stop in the solvent is the particle size.

(2) Solvent As a solvent for dispersing fine particles, an aqueous solution containing a paramagnetic ion or a paramagnetic compound and a solution of a transparent solvent other than water can be used. The concentration of the fine particles dispersed in the solvent is not particularly limited, but may be 10 ² to 10
⁷ / cm ³ is particularly suitable for the magnetophoretic concentration detection method of the present invention. Preferred combinations of the fine particles and the fine particle suspension solvent include, for example, a system in which semiconductor fine particles and environmental fine particles are dispersed in a paramagnetic solution, and a system in which magnetic fine particles are suspended in a diamagnetic solvent. In particular, the method for separating fine particles in a magnetic gradient in the present invention can be applied to a system in which biofine particles are suspended in a paramagnetic metal ion aqueous solution.
When the substance forming the fine particles is dissolved in a solvent, the same measurement and correction may be performed using a solvent saturated with the substance to perform the magnetic gradient-medium particle type magnetic trap separation method of the present invention. it can.

(3) Magnetic Fine Particles As the magnetic fine particles to which the present invention can be applied, those having a shape and a size that can be determined include polymers, ceramics,
Examples thereof include solid or gel particles such as semiconductors, blood cells, and bioparticles. The particle size of the magnetic particles is
Fine particles of the order of 0.1 micron to 100 microns are particularly suitable for the present invention.

(4) Flow Rate of Solvent The flow rate of the suspension solution that can be used in the present invention is appropriately 0.01 microliter / hour to 100 microliter / hour, and the fluctuation of the flow rate is preferably extremely small.

(5) Method and apparatus for controlling magnetic trapping force and flow rate of fine particle suspension solvent By adjusting the flow rate and / or magnetic gradient of the fine particle suspension solvent, the flowable fine particles are separated in the magnetic gradient. The flow rate of the fine particle suspension solvent can be controlled with a very small numerical value by using an appropriate fluid supply means such as a synringe pump and a syringe having a predetermined internal volume. This is because the trapping rate is proportional to the square of the radius of the particles and the magnetic gradient (max), as is clear from the equations (5) and (7). Further, the magnetic gradient is adjusted by appropriately selecting the magnetic strength and the shape of the magnet forming the magnetic field.

(6) Cell A capillary tube, a glass cell, or a plastic cell can be used as the cell, and the size of the capillary is not particularly limited. For example, the cross-section seat is 100 microns × 100 microns. preferable.

(7) Magnetophoretic velocity measuring device and experimental conditions (7-1) Magnetophoretic velocity measuring device and experimental conditions FIG. 2 schematically shows a state in which a fine particle suspension solvent is flown between a pair of magnetic poles and an observation region. Show. The magnetic trap separation state of the particles was observed using the magnetic trap separation device shown in FIG. The magnetic trap separation device includes a pair of magnets 1 having a capillary C as a cell disposed therebetween, a microscope 2 for observing magnetophoresis of fine particles, an irradiation light 3 for illuminating the capillaries, and an image obtained by the microscope. CCD camera 4 for converting the optical signal of the light into an electric signal and a monitor device 5
, And X for adjusting the position of the cell in the X and Y directions on the horizontal plane.
It comprises a Y stage 6, a syringe pump 7 for supplying the fine particle suspension solvent to the cell, a magnetic field control unit 8 for controlling the magnetic field strength formed by magnetic poles, and a supply amount control unit 9 for controlling the supply amount of the syringe pump. Reference numeral 10 indicates a magnet holder for holding a magnet, and the behavior of fine particles is determined by the microscope 2, C
Observed by the CD camera 4 and the monitor 5, recorded on the video 11 and analyzed by the personal computer 12. The cell containing the sample solution has an internal size of 100 μm.
× 100μm, outside dimension 300μm × 300μm square type capillary (Polytechnolo
ges, Square Flexible Fused
Silica Capillary Tubing)
Was used. The above-mentioned sample solution (solvent for suspending fine particles) was put into the above-mentioned capillary (length 20 cm) and placed between two magnets as shown in FIG. The magnet has a size of 16.85 mm x 1
A 9.6 mm x 2.9 mm Nd-Fe-B magnet (Sumitomo Special Metals, NEOMAX) was used. The observation region has a large magnetic field gradient from 90 μm outside the magnet end to 350 inside the magnet end.
μm. To increase the value of B (dB / dx),
The distance between the two magnets was set to 400 μm by using an aluminum spacer. The two magnets were fixed to a magnet holder made of aluminum, and the position of the magnet holder was adjusted by an XY stage.

(7-2) Measurement of magnetic susceptibility A magnetic susceptibility measuring instrument (Sherwood S
scientific LTD, MSB-AUTO) was used.

(7-3) Magnetic field analysis software "SUPERM"
Calculation of the magnetic field generated by the simulation coil or magnet by OMENT "and the generated torque is called magnetic field analysis. These calculation methods include the magnetic moment method, the finite element method, and the boundary element method.
"SUPERMOM" used for magnetic field analysis in the present invention
ENT '(H. Sekiya, 1998) is a program for the magnetic moment method, and is a product that can perform magnetic field analysis on a personal computer, which has conventionally used a large computer.

With this "SUPERMOMENT", the approximate magnetic field created by the magnet can be calculated, but the magnetic field strength around the magnet cannot be calculated exactly. The reason is that the nature and shape of the actual magnet are not completely prepared, and the influence will appear in the experimental system. In particular, in a very micro space treated in the present invention, the roundness of the ends of the magnet has a great influence on the formation of the magnetic field. However, magnetic field analysis by "SUPERMOMENT" is a very useful means for predicting a magnetic field in a small space where a magnetic field measuring instrument such as a Gauss meter cannot be used.

17 mm × for a magnet for simulation
19mm x 3mm NeFeB40 magnet (Nd-Fe above
-Same as B magnet) was used. The X-axis has a magnet end of 0, the inner direction of the magnet has a positive direction, and the Z-axis has a magnet center of 0. The distance between the magnets is 400 mm as in the experiment.

[0023]

[Examples] It was confirmed that this method enables magnetic trap separation of the polysterene fine particles used as a standard sample and the red blood cells used as an example of bioparticles.

Example 1 Polysterene particles were separated by the magnetic gradient-medium particle magnetic trap separation method according to the present invention. (1-1) Sample As the sample liquid, manganese chloride aqueous solution having μm-order polysterene latex particles dispersed therein was used as a solvent. The concentration of the manganese chloride aqueous solution was set to 0.6M.
The volume magnetic susceptibility of the manganese chloride aqueous solution at this concentration is 10
3.6 × 10 ⁻⁶ Pas, viscosity 1.12 × 10 ⁻³ Pas
s. A magnetic susceptibility measuring device (Sherw
good Scientific LTD, MSBAUT
O), a Uberroad viscometer was used to measure the viscosity. Since the density is almost the same as 1.05 gcm ⁻³ of the polysterene latex particles, the influence of the difference in density between the medium and the particles did not appear in the experiment. A particle size of 3 μm (2.7660 ± 0.1500 μm) in this 0.6 M manganese chloride aqueous solution,
6 μm (5.872 ± 0.401 μm), 10 μm
(9.14 ± 0.709 μm) of three types of polystyrene latex particles (manufactured by Funakoshi, POLYBEAD-P
OLYSTYRENE) was dispersed to a degree such that it could be easily observed individually to prepare a sample solution. These polystyrene particles are diamagnetic and their volume magnetic susceptibility is -8.21 x 10 ^-6 . The sample solution was stored refrigerated when not used, and was adjusted to room temperature (25 ° C.) during the experiment.

(2) Apparatus The test apparatus shown in FIG. 3 was used. The capillary that holds the sample solution has an inner size of 100 μm × 100 μm and an outer size of 300
Using a square type capillary of μm × 300 μm, the length of the capillary is set to 30 to 40 cm, and one end thereof is a syringe pump (Harvard Apparatus, N
ew Harvard Syringe Pumpll)
Connected to. Syringe (HAMILTON, GASTI
By using GHT # 1701) with an internal volume of 10 μl, experiments at very low flow rates of 0.0289 μlh ⁻¹ were possible. The magnet has a size of 16.85 mm
A Nd-Fe-B magnet (Sumitomo Special Metals, NEOMAX) having a size of 19.6 mm x 2.9 mm was used. The measurement area is
It was set near the magnet end (width 4 mm) where a very large magnetic field gradient is generated in the axial direction (x-axis direction) of the capillary. Since it is necessary to increase the value of B (dB / dx) in order to obtain a large trapping force, an aluminum spacer was used and the distance between the two magnets was set to 400 μm. The two magnets were fixed to a magnet holder made of aluminum, and the position of the magnet holder was adjusted by an XY stage. The behavior of the polystyrene particles was observed by a microscope (Chuo Seiki), a CCD camera (ELMO, CN42H) and a monitor, and recorded on a video for analysis.
Further, the magnet holder was arranged vertically and its position was adjusted by the XYZ stage.

(3) Trap Rate Here, the trap rate (Tra representing the efficiency of the magnetic trap is shown.
(Pipping Efficiency) is defined. As shown in FIG. 3, assuming that a range of −90 μm <x <350 μm is an observation region (Observed Region), the number of particles that have entered the observation region is N ₁ , and the number of particles that have passed through the observation region without being trapped is N _2. , The trap rate is defined as:

[Equation 6] In other words, the trap rate becomes 100% when all the particles that have entered the observation region are trapped, and the trap rate becomes 0% when all the particles pass. In the magnetic trap experiment, the trap rate with respect to the change in flow velocity was examined for each sample solution. At this time, as a measure of N ₁ , the number of trapped particles is set to a number that does not interfere with the movement of other particles that have entered the observation region.

(4) Results Table 1 summarizes the results of magnetic trap experiments of polystyrene particles of three different sizes. Further, FIG. 4 is a graph showing these results. Flow rate is 0.3 μlh
^When the flow rate is higher than ^-1, all particles are trapped, but as the flow velocity increases, smaller particles pass through, and when 6 μlh ^-1 or higher, all particles pass.

[Table 1] If the sample solution flows at the same velocity in the center of the capillary and near the wall, and if the magnetic field in the capillary is constant for a certain value of x (see Fig. 2), the trapping velocity of particles is as follows. Can be expected.

[Equation 7] That is, if the flow velocity is higher than this trapping flow velocity, it is expected that the particles will pass, and if it is lower, the particles will be trapped. What is indicated by a broken line in FIG. 4 is the trapping flow velocity predicted from the above equation. As can be seen from the graph of the results, the separation of the particles according to the size is sufficiently possible in the method for separating magnetic particles in a magnetic gradient in a magnetic gradient according to the present invention. In the magnetic separation using the flow that has been studied in the past, the magnetic force was applied in the direction perpendicular to the flow, but in the magnetic trap separation method of the present invention, the magnetic force is applied in the direction along the flow. . From this, it can be said that the magnetic trap separation method is a new analytical method that has never existed before.

[Example 2] Blood cells in blood were separated by the magnetic gradient-medium flow particulate magnetic trap separation method according to the present invention. (1) Sample As the sample liquid, one drop of blood was used in 10 ml of 0.1 M manganese chloride aqueous solution. Μm contained in blood
There are red blood cells, white blood cells, and platelets in the formed particles, which are 5.4 × 10 ⁶ in 1 mm ³ of blood,
It is included in each of 7.0 × 10 ³ and 2.5 × 10 ⁴ (average adult male). The red blood cells have a diameter of about 8.6 μm, white blood cells 10 to 20 μm, and platelets 2 to 4 μm. Although all these components are contained in the sample liquid, white blood cells are much smaller than red blood cells and platelets are small, so that red blood cells can be observed relatively easily without separation of the components. The manganese chloride aqueous solution here is 0.1M in order to make the ion concentration approximately equal to the ion concentration of physiological saline (0.155M sodium chloride aqueous solution),
Blood samples include blood anticoagulant (EDTA)
The test tube for blood cell counting attached to was used.

(2) Experimental Method An experiment was conducted with the apparatus shown in FIG. 3 using a square type capillary having an inner size of 100 μm × 100 μm as a cell. In this experiment, the size was 3 × 1 × 7 mm and the purity was 99.8.
% Iron pieces were sandwiched between Nb-Fe-B magnets, and the magnetic field gradient strength B (dB / dx) was increased to 1.800 T ² / m. At this time as well, a large magnetic field gradient is generated along the x-axis (the central axis of the capillary) of the iron piece, so the magnetic buoyancy generated in this region was used as the driving force of the magnetic trapping force. In addition, the x-axis here has a positive flow direction, and the end of the iron piece through which the flow flows is set to zero.

(3) Results Table 2 and FIG. 5 show the results of a magnetic trap experiment of red blood cells with a magnet arrangement using iron pieces. The dashed line shows the expected trapping flow rate. Red blood cells have a flow rate of 1.0 μl
All trapped below h ^-1 but 3.0 μl
When h ⁻¹ is exceeded, all pass through without being trapped, demonstrating the effectiveness of this method as a method for separating blood particles. Further, FIG. 6 shows a state in which red blood cells enter the observation region by a flow and are trapped.

[Table 2] Although the experiment is performed only on red blood cells here, the magnetic trap method seems to be effective for other components in blood.

[Brief description of drawings]

1 (a) and (b) show a pair of magnets and a capillary placed between the magnets and flowing a fine particle suspension solvent,
(C) is a graph showing the relationship between the flow direction x-axis of the capillary and the magnetic gradient formed by the pair of magnets.

FIG. 2 shows in more detail the relationship between a pair of magnets and the flow direction x-axis of a capillary in which a fine particle suspension solvent flows.

FIG. 3 schematically shows a magnetic trap separation device for particle migration velocity according to the present invention.

FIG. 4 is a diagram showing a relationship between a flow rate of a particle suspension solvent and a trap rate in a magnetic trap experiment of polyethylene particles.

FIG. 5 is a diagram showing a relationship between a flow rate of a red blood cell suspension solvent and a trap rate in a red blood cell magnetic trap experiment.

FIG. 6 shows an image captured every 3 seconds showing how red blood cells enter the observation region due to flow and are trapped in the red blood cell magnetic trap experiment.

[Simple explanation of symbols]

1 pair of magnets, 2 microscope, 3 irradiation lights, 4 C
CD camera, 5 monitors, 6 XYZ stage,
7 ... Syringe pump, 8 ... Magnetic field control unit,
9 ... Supply amount control unit, 10 ... Magnet holder, 11 ... Video tape recorder, 12 ... Personal computer (calculation device), C ... Capillary video tape recorder, 7 Calculation device (personal computer), 8
XY stage, C capillary

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI G01N 27/26 331K 331E

Claims (4)

(57) [Claims] 1. Magnetic fine particles of a plurality of sizes are suspended in a solvent, the fine particle suspension solvent is caused to flow in a predetermined direction in a minute magnetic field region, and each fine particle is directed in a direction opposite to the flow of the fine particle suspension solvent. The magnetic trapping force is caused to act by the magnetic gradient formed in the minute magnetic field region, and the magnetic trapping force acting on each fine particle and the flow velocity of the fine particle suspending solvent are controlled, so that each of them is based on the size and magnetic susceptibility of each fine particle. A method for separating magnetic particles in a liquid under a magnetic gradient for separating particles. 2. The magnetic fine particles are polymers, ceramics,
The method for separating magnetic particles in a liquid under a magnetic gradient according to claim 1, wherein the particles are solid or gel particles of semiconductors or bioparticles. 3. A pair of magnets for forming a magnetic field in a minute region, and 1
Is arranged in the minute magnetic field region formed between a pair of magnets,
A cell in which a solvent in which magnetic fine particles of a plurality of sizes are suspended is caused to flow in one direction, a trap force control means for controlling a magnetic trap force acting on each fine particle by a magnetic gradient formed in the minute magnetic field region, and the fine particles And a flow rate control means for controlling the flow rate of the suspension solvent. By controlling the magnetic trapping force acting on each fine particle and the flow rate of the fine particle suspension solvent, the fine particles are separated based on the size and magnetic susceptibility of each fine particle. , Magnetic particle separation device for magnetic particles under magnetic gradient. 4. The magnetic fine particles are polymers, ceramics,
The solid or gel-like particles of semiconductors or bioparticles according to claim 3, wherein the device for trapping magnetic particles in liquid under a magnetic gradient is used.