JPH06300754A - Coagulating or fractioning method and apparatus - Google Patents

Abstract

(57) [Summary] [Purpose] Efficient and selective quantification, separation, purification and analysis of cells, proteins and nucleic acids using magnetic beads having different Curie point temperatures. [Structure] The magnetic beads 1, 2, 3 are polystyrene beads containing a ferromagnetic material having different Curie point temperatures T1, T2, T3 (T1>T2> T3), and a secondary antibody is shared on the surface. Coated by binding. First, different primary antibodies are individually mixed and stirred with magnetic beads at a temperature higher than the Curie point temperature,
Magnetic beads labeled with primary antibody are prepared, and these three types of beads are mixed with a solution containing a sample. Then, only the analyte that specifically binds to the primary antibody of each bead specifically binds to the bead, respectively. After the cells and the beads are sufficiently bound, if the reaction solution temperature T is lowered to a temperature such that T1>T> T2,
Only magnetic beads 1 change from paramagnetic to ferromagnetic,
It has spontaneous magnetization, self-assembles, and can be easily and specifically captured by a magnet.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fractionation method and apparatus using fine particles useful for quantification, separation, purification and analysis of cells, proteins and nucleic acids. Since the fine particles of the present invention are generally formed as magnetic beads, they will be described below as magnetic beads.

[0002]

2. Description of the Related Art Binding of a sample to microparticles has been conventionally performed in order to effectively quantify and analyze a small amount of sample, and many means have been invented as means for binding microparticles to a sample. (For example, Tokuhyo 3-5042
76, Bayer, EA, & Wilchek, M .: Meth. In Bioche
m. Anal., 26, pp. 1-45 (1980)), U.S. Pat. No. 4,654.
267). Various researches have been conducted on methods for producing fine particles, and fine particles with various compositions having particle sizes of 10 nm to 10 μm have been produced (for example, Seiichiro Kashu: Surface Vol.26, No.1). , pp.27-38 (1988),
Shuichi Hamada: Surface Vol.25, No.3, pp.143-157 (1988)).

In order to quantify, analyze, and fractionate a specific sample, it is necessary to separate the target sample from other substances by some method, but the conventional centrifugation method, column separation method, electric method, etc. Methods such as electrophoresis require only a long time for separation, and there is also the possibility of denaturation or the like due to extreme changes in environmental conditions. In recent years, a method using superparamagnetic magnetic beads has been developed as a method for overcoming this disadvantage, and magnetic beads are used as a solid phase for immobilizing a sample, and proteins, cells, D
It is used for NA separation, analysis, etc.
-501958, Tokuyohei 4-501959) These superparamagnetic magnetic beads are made by dispersing magnetic substances such as iron oxide into ferromagnetic fine particles smaller than the size of magnetic domains necessary to maintain permanent magnetism in the beads. Included,
It has the property of exhibiting ferromagnetism only when an external magnetic field is applied. Utilizing this superparamagnetic property, the superparamagnetic magnetic beads are used to prevent the beads from aggregating in the solution during the reaction despite containing the magnetic substance. Only magnetic beads are aggregated specifically.

[0004]

When agglomeration of fine particles is performed by using only the conventional superparamagnetic property, it is impossible to selectively respond to an external magnetic field, and all the external magnetic fields are not responded. There is a problem that the superparamagnetic particles are aggregated. Further, if the particle size of the beads is made sufficiently small due to the property of the superparamagnetic magnetic beads, there is a problem that the force due to the magnetic field is weakened and the beads cannot be collected. In addition, since superparamagnetic magnetic beads have a property of self-assembling only in the presence of an external magnetic field, it is necessary to always act on the external magnetic field in order to move them between the containers while they are assembled.

[0005]

The above-mentioned problems can be solved by using, for the magnetic substance of the magnetic beads, a ferromagnetic substance having a Curie point temperature between −80 ° C. and 200 ° C., for example. That is, by combining a plurality of magnetic beads having different Curie point temperatures and combining a change in temperature and a magnetic field, the magnetic beads are selectively aggregated or diffused.

[0006] In addition, for magnetic beads which are desired to self-assemble,
It suffices if the beads contain ferromagnetic particles larger than the magnetic domains necessary to maintain permanent magnetism in the magnetic beads.
For magnetic beads that do not want to be self-assembled except when an external magnetic field is applied, the beads may contain ferromagnetic particles smaller than the magnetic domains necessary to maintain permanent magnetism. Regarding magnetic beads with permanent magnetism, by keeping the solution temperature above the Curie point temperature when reacting in solution,
The magnetic beads can be used in the form of being dispersed in a solution.

[0007]

[Function] Ferromagnetic material has a T
At temperatures lower than c, it becomes a ferromagnetic material and has spontaneous magnetization,
At a temperature higher than c, the paramagnetic material does not have spontaneous magnetization. Therefore, different Curie point temperatures T1, T2, T
By using beads containing a ferromagnetic material having 3, ... (T1>T2>T3> ...), in a solution in which the solution temperature is higher than T1, fine particles do not have spontaneous magnetization and diffuse in the reaction solution. Can be kept.

Next, in order to selectively separate the magnetic beads one by one, the temperature of the reaction solution is set to T1, T2, T3.
You can lower them in order and capture and separate the magnetic beads one by one.

[0009]

EXAMPLES As the ferromagnetic material for carrying out the present invention, for example, the materials shown in Table 1 below can be used.

[0010]

[Table 1]

The particle size of the magnetic beads is 10 μm.
The specific gravity is preferably 1.1 to 1.8.
The thing of is desirable.

Generally, a ferromagnetic material has a Curie point temperature Tc.
At a lower temperature, it becomes spontaneously magnetized and can be self-assembled, but at this time, an external magnetic field may be temporarily applied when the assembly is started to align the directions of the magnetic fields in the magnetic domains. Then, the fine particles will remain dispersed until an external magnetic field is applied, and after applying the external magnetic field,
It is stable as an aggregate of magnetic beads regardless of the presence or absence of a magnetic field. Also, if you do not want to aggregate except when an external magnetic field is working, disperse ferromagnetic particles smaller than the magnetic domains necessary to maintain permanent magnetism in the beads as in the conventional method. do it. In this way, the magnetic beads aggregate only when their Curie point temperature is higher than the solution temperature and an external magnetic field is applied.

Regarding the particle size of the fine particles, the moving speed of the fine particles in water becomes slower in proportion to the particle size of the fine particles. Therefore, it is preferable that the particle size be as small as possible in order to efficiently aggregate the fine particles. Also, regarding the specific gravity of the fine particles, if the specific gravity with respect to water becomes too large or too small, it cannot exist in the solution for a long enough time comparable to the reaction time, and in order to prevent aggregation, physical A mechanical stirring means becomes necessary.

Specific embodiments will be described below.

Example 1 FIG. 1 shows an example of cell separation using magnetic beads having different Curie point temperatures. The magnetic beads 1, 2, 3 are made of polystyrene containing a ferromagnetic material having different Curie point temperatures T1, T2, T3 (T1>T2> T3), and a secondary antibody is covalently coated on the surface. ing. First, different primary antibodies 4, 5, 6 at temperatures higher than the Curie point temperature, respectively.
Are individually mixed with magnetic beads and stirred to prepare primary antibody-labeled magnetic beads. Next, the primary antibody and the primary antibody-labeled magnetic beads are separated using a magnet at a temperature lower than the Curie temperature.

Next, the three types of beads are mixed with the cells 7, 8 and 9 at a temperature higher than the Curie point temperature of all the three types of magnetic beads. Then, cells 7 that specifically bind to the primary antibody 4 are beads 1, cells 8 that specifically bind to the primary antibody 5 are beads 2, and cells 9 that specifically bind to the primary antibody 6 are beads 3. Each binds specifically.

After the time required for the cells and the beads to sufficiently bond with each other, when the temperature of the reaction solution is lowered to the temperature T such that T1>T> T2, only the magnetic beads 1 are changed from the paramagnetic substance to the ferromagnetic substance. And changes. Therefore, the magnetic beads 1 have spontaneous magnetization, self-assemble as shown in FIG. 2, become heavy and sink to the bottom. In this state or by having spontaneous magnetization, it can be easily and specifically captured by a magnet.

After selectively removing the magnetic beads 1 by using a magnet, when the temperature of the reaction solution is lowered to a temperature T such that T2>T> T3, only the magnetic beads 2 change from paramagnetic substance to ferromagnetic substance. To do. Therefore, the magnetic beads 2 have spontaneous magnetization, self-assemble, and can be easily and specifically captured by the magnet.

After the magnetic beads 2 are selectively removed by using a magnet, when the temperature of the reaction solution is lowered to a temperature T where T3> T, the magnetic beads 3 are changed from paramagnetic substances to ferromagnetic substances. Therefore, the magnetic beads 3 have spontaneous magnetization, self-assemble, and can be easily captured by the magnet.

In this example, magnetic beads having different Curie points were used for cell fractionation.
NA, protein, etc. can be separated, or by combining a primer with magnetic beads, PCR can be carried out effectively and efficiently to separate them.

When beads are used for separating DNA and RNA, polystyrene beads having covalently bound streptavidin on the surface are used, and biotinylated DNA or RN is used.
A method of capturing A can also be used. To isolate and purify mRNA, magnetic beads in which 20 to 30 deoxythymidylates are covalently bonded to the surface of the beads via a 5 ′ linker may be used.

As a method for separating the self-assembled magnetic beads, in addition to a method using an external magnetic field, a centrifugation method and a filtration method using a mesh in which single beads pass but cluster beads do not pass Etc. can also be used.

Similarly, after magnetic beads and a sample are reacted in a reaction solution, liquid nitrogen (melting point -209.86) is used.
℃, boiling point -195.8 ℃, liquid ethane (melting point -18
Samples attached to magnetic beads having Curie point temperature in this temperature range in liquid diethyl ether (melting point-116.3 ° C, boiling point 34.6 ° C), etc. It is also possible to fractionate the frozen sample after freezing by instantaneously freezing and changing the solution temperature of the magnetic beads with the frozen sample dispersed in this liquid.

Example 2 FIG. 3 shows an example of separating DNA using magnetic beads having different Curie point temperatures.
The magnetic beads 10, 11, 12 are made of polystyrene having different Curie point temperatures T1, T2, T3 (T1>T2> T3), and streptavidin is covalently coated on the surface.

First, in the same procedure as in Example 1, each magnetic bead is bound to each biotin-labeled primer. Next, the magnetic beads 10, 11 and 12 with a primer are added to a solution containing DNA or RNA to be screened. After mixing well,
This mixed solution is sent to the magnetic bead fractionation device shown in FIG. Although the fractionation device is shown in a separated state in the figure, three independent regions are continuous and each region is maintained at temperatures T1, T2, and T3 by the temperature controller 15. Is dripping

First, of the magnetic beads 10, 11, 12 passing through the temperature T1 portion of the fractionation device, the magnetic beads 10 are used.
Only the magnetic beads 10 are selectively captured by the electromagnet 16 because only the paramagnetic material changes to the ferromagnetic material. Next, among the magnetic beads 11 and 12 that pass through the temperature T2 portion of the fractionation device, only the magnetic beads 11 change from a paramagnetic substance to a ferromagnetic substance, so only the magnetic beads 11 are selectively selected by the electromagnet 17. Captured by. Further, the magnetic beads 12 passing through the temperature T3 portion of the fractionation device are
Since it changes from a paramagnetic material to a ferromagnetic material, the electromagnet 18
Nucleic acids that have been captured by and that could not bind to the magnetic beads are ejected from the fractionator.

After the inside of the tube of the fractionation device is thoroughly washed with a solution containing no nucleic acid, the electromagnets 18 are gradually turned off while the extraction solution is flowing, whereby the electromagnets 18, 17, 1 are
The magnetic beads captured in 6 can be sequentially and selectively extracted.

In this example, magnetic beads having different Curie points were used for the nucleic acid fractionation.
NA, protein, cells, etc. can also be separated.

Similarly, after the magnetic beads and the sample are reacted in the reaction solution, liquid nitrogen (melting point -209.86) is used.
℃, boiling point -195.8 ℃, liquid ethane (melting point -18
Samples attached to magnetic beads having Curie point temperature in this temperature range in liquid diethyl ether (melting point-116.3 ° C, boiling point 34.6 ° C), etc. It is also possible to fractionate the frozen sample after freezing by passing the magnetic beads with the frozen sample, which are instantaneously frozen and dispersed in this liquid, through the separating device.

In each of the steps of reacting fine particles with a sample and fractionating, a liquid was used in this example, but the present invention is also applied to the reaction in the gas phase and the fractionation in the gas phase fluid in each step. Examples are readily available. Hereinafter, some of the embodiments will be exemplified.

(1) An analyte and a binding partner pair are (a) an antigen and a specific antibody, (b) a hormone and a hormone receptor, (c) a hapten and an anti-hapten,
(D) polynucleotides and complementary polynucleotides, (e) polynucleotides and polynucleotide binding proteins, (f) biotin and avidin or streptavidin, (g) enzymes and enzyme cofactors, (h) lectins and specific carbohydrates. Either. (2) In the above (1), the specimen is an antigen and the binding partner is a monoclonal antibody. (3) In the above (1), the binding partner is one or more oligonucleotide molecules. (4) In the above (1), the binding partner is one in which the oligonucleotide is covalently bound to the particle via the 5'-amino group. (5) In (1) above, the binding partner is such that the oligonucleotide has a chain length in the range of 12 to 200 bases. (6) In (1) above, the binding partner is such that the oligonucleotide is poly dT. (7) In (1) above, the binding partner is such that the oligonucleotide specifically binds to the DNA or RNA sequence of the target nucleic acid. (8) In the above (1), the binding partner has an amino group capable of forming an amide bond on the surface. (9) In (1) above, the binding partner is made of an aminoalkylated polymer in order to impart an amino group to the surface. (10) In the above (1), the binding partner has a carboxyl group capable of forming an amide bond on the surface. (11) In the above (1), the binding partner is made of a polymer or copolymer of acrylic acid in order to give a carboxyl group to the surface. (12) In the above (1), the binding partner is made of a polymer or copolymer of methacrylic acid in order to give a carboxyl group to the surface. (13) In (1) above, the binding partner has an aldehyde group capable of forming an amide bond. (14) In the above (1), the binding partner has a hydroxyl group capable of forming a covalent bond on the surface. (15) In (1) above, the binding partner is made of polyurethane with polyglycol to provide hydroxyl groups on the surface. (16) In (1) above, the binding partner is made of a cellulose derivative in order to give a hydroxyl group to the surface. (17) In the above (1), the binding partner has a silane group capable of forming a silane bond on the surface.

[0032]

INDUSTRIAL APPLICABILITY The present invention efficiently and selectively produces cells,
It is possible to quantify, separate, purify and analyze proteins, nucleic acids and the like.

[Brief description of drawings]

FIG. 1 is a flow chart showing a procedure of a first embodiment of the present invention.

FIG. 2 is a schematic diagram showing the effect of the first embodiment of the present invention.

FIG. 3 is a schematic diagram showing a second embodiment of the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Secondary antibody binding beads containing magnetic substance of Curie point temperature T2 ... Secondary antibody binding beads containing magnetic substance of Curie point temperature T2 ... 3 Secondary antibody binding beads containing magnetic substance of Curie point temperature T3 4 ... Primary antibody 1, 5 ... Primary antibody 2, 6 ... Primary antibody 3, 7 ... Target cell 1, 8 ... Target cell 2, 9 ... Target cell 3, 10 ... Beads containing magnetic substance having Curie point temperature T1 Bound sample, 11 ... Sample bound to beads containing magnetic substance at Curie point temperature T2, 12 ... Sample bound to bead containing magnetic substance at Curie point temperature T3, 13 ... Flow of solution, 14 ... Tube wall,
15 ... Heater, 16 ... Electromagnet, 17 ... Electromagnet, 18 ...
Electromagnet, 19 ... Container, 20 ... Reaction solution.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical indication location C12Q 1/06 6807-4B (72) Inventor Kenichi Kawabata 2520 Akanuma, Hatoyama-cho, Hiki-gun, Saitama Prefecture Stock Association Hitachi Research Laboratory, (72) Inventor Takeshi Fujita 2520 Akanuma, Hatoyama Town, Hiki-gun, Saitama Stock Company, Hitachi Research Laboratory

Claims (23)

[Claims] 1. A method for aggregating, which comprises binding a specimen to fine particles containing a ferromagnetic material having a predetermined Curie point temperature and controlling the temperature of the fine particles to agglomerate the specimen. 2. A different sample is bound to each of a plurality of fine particles containing a ferromagnetic material having a different Curie point temperature, and the temperature of the fine particles is controlled to fractionate different samples. Fractionation method. 3. The aggregation or fractionation method according to claim 2, wherein the ferromagnetic material has a superparamagnetic structure. 4. The fractionation method according to claim 2, wherein the surface of the fine particles has a specific binding partner for binding to each analyte to be analyzed or separated. 5. The specific gravity is in the range of 1.1 to 1.8,
The fractionation method according to claim 2 or 3. 6. The fractionation method according to claim 2, wherein the size range is less than 10 μm. 7. A first step of binding a binding partner specific to each analyte to each of a plurality of microparticles having different Curie point temperatures, and a second step of contacting each microparticle with a sample containing a plurality of analytes. A fractionation method comprising: a third step of selectively collecting fine particles while changing the temperature stepwise; and a fourth step of selectively separating the collected fine particles. 8. The first step is characterized in that one or more kinds of binding partners and fine particles having a single Curie point temperature are reacted in a container having a solution temperature higher than the Curie point temperature of the fine particles. Item 7. Fractionation method of Item 7. 9. In the second step, a plurality of types of fine particles prepared in the first step are reacted in a container containing a solution containing a sample at a solution temperature higher than the Curie point temperature of all the fine particles. The fractionation method according to claim 8, wherein 10. In the third and fourth steps, the solution temperature is changed stepwise according to the type of fine particles used,
10. The fractionation method according to claim 9, wherein fine particles to be collected in the container are selected. 11. The fractionation method according to claim 7, wherein an external magnetic field is used to select the fine particles to be aggregated in the third and fourth steps. 12. The fractionation method according to claim 7, wherein in the third and fourth steps, a centrifugation method is used to select fine particles to be collected. 13. The fractionation method according to claim 7, wherein in the third and fourth steps, a filtration method is used to select the fine particles to be aggregated. 14. The fractionation method according to claim 7, wherein in the third and fourth steps, the sample attached to the fine particles is frozen in advance and fractionated in a solution that can maintain the frozen state. 15. The fractionation method according to claim 14, wherein liquid nitrogen is used as the solution. 16. The fractionation method according to claim 14, wherein liquid ethane is used as the solution. 17. The fractionation method according to claim 14, wherein liquid diethyl ether is used as the solution. 18. The fractionation method according to claim 14, wherein liquid methane is used as the solution. 19. The fractionation method according to claim 14, wherein liquid oxygen is used as the solution. 20. The fractionation method according to claim 14, wherein liquid carbon dioxide is used as the solution. 21. A first device for binding a binding partner specific to each analyte to each of a plurality of microparticles having different Curie point temperatures, and a second device for contacting a sample containing a plurality of analytes with each microparticle. A fractionation device comprising a third device for selectively collecting the fine particles while changing the temperature stepwise and a fourth device for selectively separating the collected fine particles. 22. A third and a fourth device are a set of a temperature control device and a magnetization control device, and a plurality of sets are independently set along a pipe through which each fine particle specifically bound to a plurality of analytes flows. 22. The fractionation device according to claim 21, wherein the fractionation device is arranged as a unit. 23. A first device for binding a binding partner specific to an analyte to each of a plurality of microparticles having a predetermined Curie point temperature, and a second device for contacting a sample containing a plurality of analytes with each microparticle. An aggregating apparatus comprising a third device for collecting fine particles by selectively changing the temperature.