JP2005295868A - Magnetic bead and isolation method of specimen using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide magnetic beads deflocculating specimens at a low temperature causing no degeneration nor destruction and capable of preventing degeneration and destruction of the specimens, and to provide a method for isolating the specimens by using the beads. <P>SOLUTION: The method comprises mixing, in a sample 14 containing a several kind of specimens 11, 12 and 13, a several kind of magnetic beads 1B1, 1B2 and 1B3 each covered with a nonmagnetic coat specifically bonding or reacting with the several kind of specimens on the surface of magnetic particles comprising ferrimagnets having compensation temperatures in each different temperature ranges, specifically bonding or reacting each specimen with each magnetic bead, then, being influenced with an external magnetic field to agglomerate superparamagnetic beads in the external magnetic field, adjusting the temperature of the mixture of the sample and the beads at the compensation temperature of the magnetic beads 1 to deflocculate the agglomerated magnetic beads 1 and taking out, under such a condition, the magnetic beads 1 and the specimens specifically bonded or reacted therewith. The steps are repeated required number of repetitions. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a magnetic bead useful for quantification, separation, purification and analysis of specimens such as cells, proteins and nucleic acids, and a specimen separation method using the same.

  In order to quantify, analyze, or fractionate a specific specimen, it is necessary to separate the target specimen from other substances and take it out. Conventionally, centrifugal separation, column separation, electrophoresis and the like have been widely used as means for separating a target specimen from other substances, but these means all require a large amount of specimen. Moreover, not only does it take a long time to separate the target sample, but there is also a disadvantage that the sample is easily denatured because the environmental conditions of the sample are extremely changed.

  In recent years, a method using superparamagnetic magnetic beads has been developed as a technique that can overcome the disadvantages of the conventional method, and is used for separation and analysis of specimens such as cells, proteins, DNA, and RNA (for example, patent documents). 1).

  Superparamagnetic beads are dispersed in non-magnetic beads that specifically bind to or react with a specific analyte, such as iron oxide, which is finer than the size of the magnetic domain necessary to maintain permanent magnetism. It has the property of exhibiting ferromagnetism only when an external magnetic field is applied. Therefore, this superparamagnetic magnetic bead is put into a sample containing a target specimen without applying an external magnetic field, and the target specimen is bound to the surface of the bead, and then an external magnetic field is applied to superparamagnetic magnetic beads. Can be collected together with superparamagnetic magnetic beads.

  In addition, as a method for individually separating a plurality of specimens contained in a sample, the combination of the Curie temperature of the magnetic material and the binding partner that specifically binds or reacts with a specific specimen modified on the surface of the magnetic beads is different. Put multiple types of superparamagnetic beads into a sample containing multiple specimens, individually bind the target specimen to the surface of each superparamagnetic magnetic bead, and then apply an external magnetic field to all superparamagnetic magnetic beads. Then, the sample temperature is raised to the Curie temperature of the magnetic substance contained in the superparamagnetic magnetic bead 1 to selectively unaggregate the superparamagnetic magnetic bead 1 and There has also been proposed a method of repeating the process of recovering one superparamagnetic magnetic bead released from the sample together with a specimen bound thereto (see, for example, Patent Document 2).

According to this method, a plurality of specimens contained in one sample can be sequentially separated individually by raising the temperature of the sample stepwise to the Curie temperature of the magnetic substance contained in each superparamagnetic magnetic bead. Therefore, it is possible to efficiently separate a plurality of target specimens from one sample.
Japanese National Patent Publication No. 4-501956 JP-A-6-300754

  However, since all of the currently known magnetic materials have a Curie temperature of 100 ° C. or higher, for example, the function of certain antibodies may be destroyed in the process of separation, and can be applied. There is a problem that the types of specimens are limited.

  The present invention has been made to solve such deficiencies of the prior art, and an object of the present invention is to provide magnetic beads capable of releasing aggregation at a temperature at which the specimen is not denatured or destroyed, and a specimen separation method using the same. And to provide.

  In order to solve the above problems, the present invention relates to a magnetic bead comprising a magnetic particle and a nonmagnetic coating layer coated on the surface of the magnetic particle, wherein the magnetic particle has a compensation temperature. It consists of a ferrimagnetic material.

  As the ferrimagnetic material, it is particularly preferable to use a rare earth element-transition metal element alloy because the compensation temperature can be easily controlled by the composition of the magnetic material. In addition, since the coating layer has a high function of preventing aggregation of the magnetic substance and can selectively bind a binding partner (probe) that specifically binds or reacts with a specific specimen, silica, It is particularly preferable to use one made of any one material selected from alumina, apatite, and chitosan. Furthermore, a probe made of a functional group or the like that specifically binds to a functional group of the specimen can be provided on the surface of the coating layer so that more specimen can be specifically bound or reacted. Examples of the probe include a specific antigen that specifically binds or reacts with a specific antigen, a hormone receptor that specifically binds or reacts with a specific hormone, an anti-hapten that specifically binds or reacts with a specific hapten, A complementary polynucleotide that specifically binds or reacts with a specific polynucleotide, a polynucleotide-binding protein that specifically binds or reacts with a specific polynucleotide, an avidin or a strep that specifically binds or reacts with a specific biotin Examples include avidin, an enzyme cofactor that specifically binds or reacts with a specific enzyme, and a specific hydrocarbon that specifically binds or reacts with a specific lectin.

  On the other hand, as a method for separating specimens using magnetic beads, first, in the specimen containing plural kinds of specimens, the plural kinds of specimens are respectively formed on the surfaces of magnetic particles made of ferrimagnetic materials having different compensation temperatures. A plurality of types of magnetic beads formed by coating a nonmagnetic coating layer that specifically binds or reacts with one of the plurality of samples, and the plurality of types of specimens and the coating layer of the plurality of types of magnetic beads are each specifically After binding or reacting, an external magnetic field is applied to aggregate all the magnetic beads into an external magnetic field, and then the temperature of the mixture of the sample and the magnetic beads is set to the compensation temperature of the plurality of types of magnetic particles. Each time the temperature of the mixture of the sample and the magnetic beads reaches the compensation temperature of one type of magnetic particles, the magnetic beads having the one type of magnetic particles and the specific binding or binding thereto. The test that reacted Was the configuration that selectively take out the door.

  Second, in a sample containing a plurality of types of analytes, probes that specifically bind to or react with one of the plurality of types of analytes on the surface of a magnetic particle made of a ferrimagnetic material having different compensation temperatures. A plurality of types of magnetic beads coated with a nonmagnetic coating layer are mixed, the plurality of types of specimens and the probes of the plurality of types of magnetic beads are respectively specifically bound or reacted, and then an external magnetic field is applied. All the magnetic beads are aggregated in an external magnetic field, and then the temperature of the mixture of the sample and the magnetic beads is adjusted stepwise to the compensation temperature of the plurality of types of magnetic particles, and the sample and the magnetic Each time the temperature of the mixture with the beads reaches the compensation temperature of one type of magnetic particles, the magnetic beads having the one type of magnetic particles and the sample that specifically binds to or reacts with the magnetic beads are selectively extracted. When It was in the jar configuration.

  Ferrimagnetic materials have magnetic atoms on two or more sets of crystal sublattices, and when the magnetic moments A and B of the magnetic atoms are opposite to each other as shown in FIG. Spontaneous magnetization corresponding to the difference between the magnetic moments A and B occurs. This spontaneous magnetization has temperature dependence, and depending on the composition, as shown in FIG. 6, there is a ferrimagnetic material that does not exhibit spontaneous magnetization at a temperature Tcomp that is lower than the Curie temperature Tc. This temperature Tcomp that does not exhibit spontaneous magnetization is called “compensation temperature”, and has spontaneous magnetization at a temperature higher or lower than the compensation temperature. For this reason, when multiple types of magnetic beads with different compensation temperatures are mixed in the sample, if the sample temperature is not the compensation temperature of any of the magnetic beads, an external magnetic field is applied to make all the magnetic beads an external magnetic field. When the temperature of the sample is adjusted to the compensation temperature of one magnetic bead, the aggregation due to the external magnetic field of the one magnetic bead can be released.

  Therefore, a plurality of types of magnetic beads having different combinations of the compensation temperature of the magnetic material and the sample to which the non-magnetic material specifically binds or reacts are put into a sample containing a plurality of samples, and the target sample is applied to each magnetic bead. After the individual beads are bonded, the temperature of the sample is raised to the compensation temperature of the magnetic material constituting the magnetic beads of 1 to deaggregate the magnetic beads of 1 and the magnetic beads having been deaggregated are bonded to them. By repeating the process of collecting the sample together with the sample as many times as necessary, a plurality of target samples are individually separated from one sample at a lower temperature than when multiple types of superparamagnetic magnetic beads having different Curie temperatures are used. be able to. Therefore, inconveniences such as denaturation and destruction do not easily occur in the specimen, the target specimen can be separated with high efficiency, and the application range of the magnetic beads can be expanded.

  According to the present invention, since the magnetic particles are made of a ferrimagnetic material having a compensation temperature, a specimen that specifically binds or reacts with the compensation temperature when separately separating a plurality of target specimens from one sample, By putting a plurality of types of magnetic beads having different combinations into a sample containing a plurality of specimens, a plurality of types of superparamagnetic magnetic beads having different Curie temperatures can be used at a lower temperature than a plurality of purposes. The specimens to be separated can be individually separated, so that the efficiency of specimen separation can be improved and the application range of magnetic beads can be expanded.

  Hereinafter, an embodiment of a magnetic bead according to the present invention will be described with reference to FIGS. FIG. 1 is a cross-sectional view showing a first example of a magnetic bead according to the present invention, FIG. 2 is a cross-sectional view showing a second example of the magnetic bead according to the present invention, and FIG. 3 is various figures applicable to the magnetic bead according to the present invention. It is a table | surface figure which shows this ferrimagnet, and its compensation temperature.

  As shown in FIG. 1, the magnetic bead 1 </ b> A according to the first example includes a magnetic particle 2 and a nonmagnetic coating layer 3 coated on the surface of the magnetic particle 2.

The magnetic particle 2 is formed of a ferrimagnetic material having a compensation temperature. Any ferrimagnetic material having a compensation temperature can be used as long as it has a compensation temperature. However, since the compensation temperature is relatively close to room temperature, a rare earth element-transition metal element based alloy should be used. Is particularly preferred. FIG. 3 shows various ferrimagnetic materials applicable as the magnetic particles 2 and their compensation temperatures. As is apparent from this _figure, the compensation temperature of _{3Dy 2 O 3 · 5Fe 2 O} 3 is -40 ° _C., the compensation temperature of _{3Tb 2 O 3 · 5Fe 2 O} 3 is _{-30 ℃, 3Gd 2 O 3 ·} 5Fe 2 O ₃ is 0 ° C., Y ₃ Ga ₂ Fe ₃ O ₁₂ is −130 ° C., LiCr ferrite is room temperature to + 100 ° C., GdFe, GdTbFe, and GdDyFe are compensation temperatures of −100 ° C. to + 100 ° C., respectively. ° C.

  The coating layer 3 is a non-magnetic material that specifically binds or reacts with a specific specimen, a tosyl group that fixes a chemical substance having an epoxy group at a linker end having a specific functional group, such as an amino group or a thiol group, It is formed with an activated carboxyl group that fixes a chemical substance having a hydroxyl group, an amino group that fixes a chemical substance having a carboxyl group, and a nonmagnetic substance substituted with a thiol group that fixes a chemical substance having a promoacetamide group. As the non-magnetic substance that specifically binds or reacts with the specific analyte, it has a high function of preventing aggregation of the magnetic substance and allows selective binding of the probe, so silica, alumina, apatite, or chitosan. Etc. can be used.

  On the other hand, the magnetic beads 1B according to the second example are bonded to the magnetic particles 2, the nonmagnetic coating layer 3 coated on the surface of the magnetic particles 2, and the nonmagnetic coating layer 3 as shown in FIG. The probe 4 is made up of. Examples of the probe 4 include a specific antigen that specifically binds or reacts with a specific antigen, a hormone receptor that specifically binds or reacts with a specific hormone, an anti-hapten that specifically binds or reacts with a specific hapten, A complementary polynucleotide that specifically binds or reacts with a specific polynucleotide, a polynucleotide-binding protein that specifically binds or reacts with a specific polynucleotide, an avidin or strept that specifically binds or reacts with a specific biotin Examples include avidin, an enzyme cofactor that specifically binds or reacts with a specific enzyme, and a specific hydrocarbon that specifically binds or reacts with a specific lectin. Since others are the same as the magnetic bead 1A according to the first example, description thereof is omitted.

  Next, taking as an example the case of using the magnetic beads 1B according to the second example, a method for individually separating a plurality of target specimens from one sample will be described based on FIG. FIG. 4 is an explanatory diagram showing an example of a specimen separation method according to the present invention.

  First, as shown in FIG. 4A, a magnetic bead 1B1 having a magnetic particle 2a having a compensation temperature T1 and a first probe 4a, a magnetic particle 2b having a compensation temperature T2 and a second probe 4b are provided. And magnetic beads 1B3 having magnetic particles 2c having a compensation temperature of T3 and third probes 4c.

  Next, as shown in FIG. 4 (b), these magnetic beads 1B1, 1B2, and 1B3 are put into a sample 14 containing at least three types of specimens 11, 12, and 13, and stirred first. The specimens 11, 12, and 13 are individually coupled to the third to fourth probes 4a, 4b, and 4c, respectively. Samples 11, 12, and 13 can be cells, proteins, nucleic acids, or other chemical substances. The sample 14 may be any of a liquid phase, a gas phase, and a gas phase fluid.

  Next, as shown in FIG. 4C, while applying an external magnetic field to the sample 14 using the magnet 15, the temperature of the sample 14 is adjusted to T1 which is the compensation temperature of the magnetic particles 2a using the temperature controller 16. To do. As a result, only the magnetic beads 1B1 that do not exhibit spontaneous magnetization are selectively precipitated on the bottom of the sample 14, and the magnetic beads 1B2 and 1B3 that exhibit spontaneous magnetization are aggregated in the magnet 15. As the magnet 15, an electromagnet can be used, or a permanent magnet can be used. Further, as the temperature controller 16, a Peltier element or the like can be used.

  Therefore, as shown in FIG. 4 (d), the magnetic beads 1B1 and the specimen 11 bound to the magnetic beads 1B1 are isolated by removing the magnetic beads 1B1 from the bottom of the container containing the sample 14 and storing them in another container. Can be taken out with.

  Next, as shown in FIG. 4E, while applying an external magnetic field to the sample 14 using the magnet 15, the temperature of the sample 14 is adjusted to T2 which is the compensation temperature of the magnetic particles 2b using the temperature controller 16. . As a result, only the magnetic beads 1B2 that do not exhibit spontaneous magnetization selectively precipitate on the bottom of the sample 14, and the magnetic beads 1B3 that exhibit spontaneous magnetization are aggregated in the magnet 15.

  Therefore, as shown in FIG. 4 (f), the magnetic beads 1B2 are extracted from the bottom of the container containing the sample 14 and stored in another container, so that the magnetic beads 1B2 and the specimen 12 bound to the magnetic beads 1B2 are separated. Can be taken out with.

  Finally, as shown in FIG. 4G, the temperature of the sample 14 is adjusted to T3, which is the compensation temperature of the magnetic particles 2c, using the temperature controller 16. As a result, the magnetic beads 1B3 that do not exhibit spontaneous magnetization are deposited on the bottom of the sample 14.

  Therefore, as shown in FIG. 4 (h), the magnetic beads 1B3 and the specimen 13 bound to the magnetic beads 1B3 are isolated by removing the magnetic beads 1B3 from the bottom of the container containing the sample 14 and storing them in another container. Can be taken out with.

  As described above, since the compensation temperature of the ferrimagnetic material is much lower than the Curie temperature, the magnetic beads 1B according to the present invention are used to individually separate a plurality of specimens from one sample by the above method. Then, inconveniences such as denaturation and destruction do not easily occur in the specimen, the target specimen can be separated with high efficiency, and the application range of the magnetic beads can be expanded.

  In the above description, the magnetic beads 1B1, 1B2, and 1B3 having the probes 4a, 4b, and 4c are used. However, when the magnetic beads 1A that do not have the probes 4a, 4b, and 4c are used, the sample is used in the same manner. Separation can be performed. When the separation of DNA or RNA is performed using magnetic beads whose surface is coated with silica, a specific DNA probe or RNA probe on the surface of silica may be modified and used for hybridization. In addition, when mRNA is isolated and purified, 20 to 30 deoxythymidylic acid covalently bound to the surface of the magnetic beads via a 5 ′ linker can be used.

  In the above description, the magnetic beads 1B1, 1B2, and 1B3 are used for the separation of the specimen. However, by combining the primers with the magnetic beads 1B1, 1B2, and 1B3, PCR can be effectively and efficiently separated. Is possible.

  Hereinafter, other examples of the magnetic beads according to the present invention will be listed.

(1) A monoclonal antibody is used as a binding partner of an antigen as a specimen.

(2) One or more oligonucleotide molecules are used as the binding partner of the antigen as the specimen.

(3) It is assumed that an oligonucleotide is covalently bound to a magnetic bead via a 5′-amino group as a binding partner of an antigen as a specimen.

(4) It is assumed that the oligonucleotide has a chain length in the range of 12 to 200 bases as a binding partner of an antigen as a specimen.

(5) Assume that the binding partner of the antigen as the specimen is the oligonucleotide dT.

(6) The binding partner of the antigen as the specimen is specifically bound by the oligonucleotide to the DNA or RNA sequence of the target nucleic acid.

(7) A binding partner of an antigen as a specimen has an amino group capable of performing amino binding on the surface.

(8) A binding partner of an antigen as a specimen is made of an aminoalkylated polymer in order to give an amino group to the surface.

(9) A binding partner of an antigen as a specimen has a carboxyl group capable of amino bonding on the surface.

(10) A binding partner of an antigen as a specimen is made of an acrylic acid polymer or copolymer in order to give a carboxyl group to the surface.

(11) A binding partner of an antigen as a specimen is made of a polymer or copolymer of methacrylic acid in order to give a carboxyl group to the surface.

(12) A binding partner of an antigen as a specimen has an aldehyde group capable of amino binding.

(13) A binding partner of an antigen as a specimen has a hydroxyl group capable of covalently binding to the surface.

(14) The binding partner of the antigen as the specimen is made of polyurethane with polyglycol to give a hydroxyl group on the surface.

(15) A binding partner of an antigen as a specimen is made of a cellulose derivative in order to give a hydroxyl group to the surface.

(16) A binding partner of an antigen as a specimen has a silane group capable of silane bonding on the surface.

  In addition, in the sample separation method, a method of putting a plurality of types of magnetic beads into a sample has been described. However, the magnetic beads according to the present invention are not limited to such a method of use, and are conventional superparamagnetic. As with the magnetic beads, it can be applied to the separation of the specimen alone.

It is sectional drawing which shows the 1st example of the magnetic bead which concerns on this invention. It is sectional drawing which shows the 2nd example of the magnetic bead which concerns on this invention. It is a table | surface figure which shows the various ferrimagnetic bodies applicable to the magnetic bead which concerns on this invention, and its compensation temperature. It is explanatory drawing which shows an example of the separation method of the sample which concerns on this invention. It is explanatory drawing which shows the magnetic moment of a ferrimagnetic body. It is a graph which shows the temperature dependence of the spontaneous magnetization of a ferrimagnetic material.

Explanation of symbols

1A, 1B Magnetic beads 2 Magnetic particles 3 Coating layer 4 Probe 11, 12, 13 Specimen 14 Sample 15 Magnet (external magnetic field)
16 Temperature controller

Claims (7)

  A magnetic bead comprising magnetic particles and a nonmagnetic coating layer coated on the surface of the magnetic particles, wherein the magnetic particles are made of a ferrimagnetic material having a compensation temperature.   The magnetic bead according to claim 1, wherein the ferrimagnetic material is made of an alloy containing at least a rare earth element and a transition metal element.   The magnetic bead according to claim 1, wherein the coating layer is made of any one material selected from silica, alumina, apatite, and chitosan.   The magnetic bead according to claim 1, further comprising a probe that specifically binds or reacts with a specific specimen on the surface of the coating layer.   A specific antigen that specifically binds or reacts with a specific antigen, a hormone receptor that specifically binds or reacts with a specific hormone, an anti-hapten that specifically binds or reacts with a specific hapten, a specific A complementary polynucleotide that specifically binds or reacts with a polynucleotide, a polynucleotide-binding protein that specifically binds or reacts with a specific polynucleotide, avidin or streptavidin that specifically binds or reacts with a specific biotin, The enzyme cofactor that specifically binds or reacts with a specific enzyme, and a specific hydrocarbon that specifically binds or reacts with a specific lectin. 4. Magnetic beads according to 4.   A sample containing a plurality of types of specimens is coated with a nonmagnetic coating layer that specifically binds or reacts with one of the plurality of kinds of specimens on the surface of a magnetic particle made of a ferrimagnetic material having different compensation temperatures. A plurality of kinds of magnetic beads are mixed, and the plurality of kinds of specimens and the coating layers of the plurality of kinds of magnetic beads are respectively specifically bound or reacted, and then an external magnetic field is applied to all the magnetic beads. The temperature of the mixture of the sample and the magnetic beads is adjusted stepwise to the compensation temperature of the magnetic particles, and the temperature of the mixture of the sample and the magnetic beads is increased. Separation of specimen characterized by selectively taking out the magnetic beads having the one kind of magnetic particles and the specimen specifically bound or reacted with the magnetic beads each time the compensation temperature of one kind of magnetic particles is reached. Method.   A nonmagnetic coating layer having a probe that specifically binds or reacts with one of the plurality of analytes on the surface of a magnetic particle made of a ferrimagnetic material having different compensation temperatures in a sample including the plurality of analytes A plurality of types of magnetic beads coated with a plurality of types of magnetic beads are mixed, and the plurality of types of specimens and the probes of the plurality of types of magnetic beads are respectively specifically bound or reacted, and then an external magnetic field is applied to all the magnetic fields. The beads are aggregated in an external magnetic field, and then the temperature of the mixture of the sample and the magnetic beads is adjusted stepwise to the compensation temperature of the plurality of types of magnetic particles, and the mixture of the sample and the magnetic beads is adjusted. Each time the temperature reaches the compensation temperature of one kind of magnetic particles, the magnetic beads having the one kind of magnetic particles and the specimen that specifically binds or reacts to the magnetic beads are selectively taken out. Body method of separation.

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