The method of separation of magnetic microparticles with their pre-concentration and device for its implementation

 

(57) Abstract:

The invention is intended for separating magnetic particles from a mixture containing them. First carry out the concentration of particles on the wall of the vessel containing the mixture, and then use magnetic media having a protective housing and a magnet movable relative to the protective housing, collect particles with the walls of the vessel. The invention effectively when it is necessary to collect particles from a rather large volume and transfer to a significantly smaller size. 2 C. and 8 C.p. f-crystals, 10 ill.

The invention relates to the separation of magnetic particles from the containing mixture. The invention may have various applications, particularly in the fields of biotechnology, biochemistry and Biomedicine.

Magnetic microparticles are widely used for binding the biomaterial. One of the advantages of microparticles is a large surface area of the solid phase and the small diffusion length. The size of the microparticles is typically 0.05 - 10 μm, they can be manufactured from various materials and have found various applications. Magnetic particles can be moved by means of a magnet.

Commonly used methods of separating magnetic particles predusmatrivaetsya. After that, the liquid that is released from the particles, is decanted or sucked. However, removal of the fluid from the vessel must be very careful not to delete and particles.

In European application EP-140787 (corresponding to U.S. patent 4649116) proposed a method, according to which magnetic microparticles are separated from the liquid by using the insertion into it of the magnetic core. Particles are separated from the rod by using a stronger magnet.

In the international application WO-86/06493 proposed method, intended for use in immunological analysis, according to which the magnetic particles and the associated marked complex is separated from the liquid using a rod magnet and then selected for measurements. At the end of the rod are fixed magnet and a removable protective housing, on the outer surface of which particles are held. After separation of the particles and before the measurement, it is desirable to close the protective case is another case. After measuring both hulls are removed together with the particles are thrown out and replaced with new buildings for the next particle separation. According to the application, as the mV international application WO-87/05536 a device for separating magnetic particles, inside which there is a rod that is movable in a vertical hole, and a magnet at its lower end. The device is introduced into the liquid containing the particles, when the magnet is in the lower position, causing the particles accumulate at the end of the rod. When the magnet means in the upper position, allows the separation of particles from the terminal. This makes it possible to collect particles and transfer them from one fluid to another.

However, the described devices and methods for separating magnetic particles can be used effectively in cases where it is necessary to collect particles from a rather large volume and transfer to a significantly smaller size.

The invention is directed to solving tasks easy and efficient collection of magnetic particles contained within the vessel is quite a large amount.

For this task offers a new method of particle separation and a new device for carrying out the method. There are also preferred embodiments of the invention.

According to the present invention in the separation of particles from a mixture of first particles concentrate in one area of the vessel where they are, and then collect with COI/P> Concentration can be carried out using a magnetic field, gravity or centrifugation. In most cases it is preferable to use a magnet.

The collection of the particles preferably is carried out using the funds for the Department in the form of an elongated body, movably installed in the housing and provided with a magnet attached to its lower end and creating a magnetic field. It is preferable to use a longitudinally oriented magnetic field intensity and gradient which reaches the highest values near the lower end of the means for separating. Thanks particles accumulate, concentrating at the end of the tool, where they can be transferred to a small volume. In addition, using a similar tool to separate the particles, it is easy to ensure a high concentration of particles in the process of collection.

The localization of the magnetic field at the end of the means for separating particles preferably be provided by a long rod of the magnetic core. The ratio of the length of the magnetic core to its thickness is at least 2:1, preferably at least 3:1 and most preferably at least 12:1. Preferably, this rod had destacamos composition.

The magnetic core preferably consists of a permanent magnet and a ferromagnetic rod attached to the upper end of the magnet and which is its continuation. Due to this, the magnet and the magnetized rod together act as a long bar magnet. The rod reduces the gradient at the upper pole of the magnet, so that the upper pole does not collect particles. This way you can get the long bar magnet at low cost. However, even in the presence of a ferromagnetic rod is preferable to use relatively long magnet (the length of which is 2-10 times greater than its thickness). It is preferable to choose such a length of the magnet to provide the maximum intensity of the internal field constant. The junction between the magnet and the rod is preferably performed so that in a short length of the rod and the magnet included in each other. The result eliminates the formation of strong gradients in the joint area, which could lead to the collection of particles.

The cross-section of a rod magnet can be, for example, round or rectangular. The round shape is the best from the point of view of manufacture and application. Indeed, in this case, the rotation of the magnet around its t to be made curved.

Removing particles from the surface of the means for separating may be performed in any suitable way, for example, mechanically or using another magnet. However, preferably the particulate removal is performed by removing the magnetic field generated by the means for separating particles.

The extremity of the protective housing is preferably provided with a downward directed arrow. This minimizes the amount of liquid remaining in the tip casing. Typical shape of the tip is a cone. When migrating particles in very small vessels extremity preferably as a cone with a concave surface.

The shape of the protective housing on the rod depends on the specific application. As a rule, from the standpoint of manufacture and use, the most convenient round shape. To increase the strength of the case you can do the taper, which will also simplify the manufacture of the body by the method of injection molding.

This method can be applied with simple hand-held devices, and automatic multi-channel systems.

The invention is most advantageously used for particle sizes of 1-10 μm.

Further, as examples, b is a:

in Fig. 1 is a means for separating particles, useful for carrying out the invention

in Fig. 2 - the concentration of particles on the wall of the tubes;

in Fig. 3 - collection of particles with the walls of the tubes;

in Fig. 4 - collecting particles from the bottom of the tubes;

in Fig. 5 is another variant of the collection of particles with the walls of the tubes;

in Fig. 6 is a device for the separation and transfer of particles, which is useful for carrying out the invention at the stage of concentration of the particles;

in Fig. 7 - device according to Fig. 6 at the stage of collecting particles;

in Fig. 8 - separation of particles on the bottom of the elongated tray;

in Fig. 9 - the collection of particles from the tray according to Fig. 8; and

in Fig. 10 is a vessel for separating particles at the bottom of which made several grooves for separating particles.

Means for separating particles according to Fig. 1 includes an elongated protective housing 1 in which there is a channel 2. The lower ends of the housing 1 and channel 2 is made slightly tapering. So it's easier to keep on the top end of the housing 2 has a flange 3.

Channel 2 is freely magnetic rod 4. It consists of a vertical rod magnet 5 in the lower part thereof and located above him ferromagnetic rod 6, is made as a continuation of the rod magnet. On U 8 with a concave surface. The length of this tip approximately corresponds to the width of the lower end of the housing.

The ratio of the length of the magnet 5 and its diameter is about 10:1 and the ratio of the length of the rod to the length of the magnet is about 5:1. The stem is slightly thicker than the magnet, and the upper end of the magnet enters the lower end of the rod to a length about twice the diameter of the magnet.

Tip 8 is particularly well adapted for transport of the particles in very small vessels type cells 11 in the so-called HLA-Board (Board for research with human leukocyte antigen). The length of the tip in this case is slightly higher than the height of the cell. When the tip is introduced into the cell, the surface of the liquid due to the action of surface tension, rises on the surface of the tip. The edge of the moving surface of the liquid washes away particles from the tip in the liquid. Separation of particles from the tip can be enhanced by vzbaltyvaya movements of the rod. When the tip is removed from the cell, the liquid moves toward the edge of the tip as a single film. Because of this fluid with particles in it completely separates from the tip.

In Fig. 2 and 3 shows how the particles first prithivi the odya on her terminal device for separating, with the formation of mass 10 in the form of spots. The width of the magnet 11 coincides with the height of the liquid in the test tube, and the magnet is magnetized in the direction of its short side. The extremity of the protective housing 1 in this case has the form of a short cone with a relatively large angle at the vertex. This version is adapted for transport of the particles in the vessels when the end can be freely introduced into the liquid. In the rest of the rod 1 is similar to the rod,

it is shown in Fig. 1.

The method, which is illustrated in Fig. 2 and 3, was tested in the transfer of particles from the tube type Vacutainer (volume 7 ml) in a cell card, micrometrology (50 μl). The concentration of particles was carried out NdFeB-magnet dimensions HH mm Separating the rod contained NdFeB-magnet with a diameter of 3 mm and a length of 2 mm. In the preliminary tests, the time spent on collecting particles was about 1 min.

In accordance with one variant of the invention uses a vertical tube, for example a V-shaped groove on the wall. Particles deposited in the groove with education may narrow strips. This also facilitated the collection of particles by moving means for separating the bottom of the groove.

Fig. 4 illustrates the collection of particles is preferably by use of gravity or centrifugation. Means for separating similarly shown in Fig. 3.

Fig. 5 illustrates a collection of particles with the wall of the tube using another variant, the means for separating. It also draws on the movable rod 4' in channel 2 of the protective casing 1'. Its tip has a small magnet 5', while the rod 6' is not magnetic. The magnet is oriented in the transverse direction relative to the rod length. In this case, particles are accumulated on opposite sides of the tip with mass formation 10.

In Fig. 6 and 7 presents a multichannel automatic device for implementing the method. The device used the funds for the Department according to Fig. 5. Particles are collected on the wall of the tube under the action of bar magnets 11, mounted in the housing of the device near its lateral surface. Tubes of which must be allocated to the particles, are installed along the edge of the carriage 12. The carriage can move relative to the housing so that the tubes are brought to the magnets, resulting in the concentration of particles on the walls of the tubes. Then the carriage away from the magnets and the particles are collected from the walls by the means for separating (dividing with Osada particles. Tip of the separating rods with being on particles down to the appropriate cells, after which the magnetic cores are picked up and released particles are transported into the cell.

Fig. 8 and 9 illustrate the separation of particles in an elongated tray, the cross section of which is made tapering down. Using the magnet 11.1 particles are attracted to the bottom of the tray with the formation of elongated horizontal line 9.1. Then the magnet and push the particles are collected using a means for separating particles. In this case, the effect on the particle is in the direction of their natural movement during deposition in a relatively shallow layer of liquid. The Department is fast, the line particles localised, and the collection of particles is easy. Preferably at the bottom of the tray V-shaped groove, in which the accumulated particles in the form of possible narrow line that ultimately facilitates their collection.

Variant according to Fig. 10 includes a vessel for separating particles, in which you have many parallel grooves of V-shaped cross section, respectively, under the bottom of the vessel placed many magnets 11.1. Particles accumulate, forming a narrow line 9.2 at the bottom of the grooves. The liquid surface lay the skill sets of transverse channels, to ensure a constant level of liquid in all the grooves.

1. The method of separating magnetic particles from the mixture in the vessel, including the collection of particles using magnetic means to separate, which hold the particles and subsequent removal of the particles from the specified magnetic media by removing the magnetic field created by the specified magnetic means, characterized in that prior to particle collection exercise their concentration at the vessel wall and the particle collection using the specified magnetic media produced in this zone.

2. The method according to p. 1, characterized in that the concentration of particles is performed with the use of magnetic fields, gravitation or centrifugation.

3. The method according to p. 2, characterized in that the concentration of particles is performed with the use of a magnetic field.

4. The method according to any of paragraphs.1 to 3, characterized in that the concentration of particles is carried out on the side of the vessel with the formation of vertical stripes.

5. The method according to any of paragraphs.1 to 3, characterized in that the concentration of particles is carried out with the formation of horizontal stripes on the bottom of the vessel.

6. The method according to p. 4 or 5,/P> 7. Device for separating magnetic particles from a mixture containing particles containing vessel to mix and magnetic means for separating particles, provided with a protective casing and a magnet, made movable relative to the protective housing, characterized in that it further provided with means for concentrating the particles on the wall of the vessel for the collection of particles in this zone.

8. The device according to p. 7, characterized in that the means for separating includes an elongated body with an upper end and a lower end movable in a longitudinal voltage relative to the protective housing, and the magnet is fixed at the lower end of the elongated body.

9. The device under item 7 or 8, characterized in that the means for separating includes magnetic rod, oriented in the direction of the longitudinal axis of the elongated body, the length of the magnetic core to its thickness is chosen not less 2 : 1, preferably not less 3 : 1 and especially preferably not less 12 : 1.

10. The device according to p. 9, characterized in that the magnetic core contains a magnet at its lower end and a ferromagnetic rod attached to the upper end of the magnet.

 

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