JP2006334562A - Dispersion of magnetic material particle having metal chelate compound on surface - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a metal chelate compound to be used as a carrier matrix suitable for metal chelate affinity chromatography or a magnetic material particle, a method for producing the metal chelate compound and an inexpensive metal chelate resin having extremely large bonding capacity. <P>SOLUTION: Sylylpropylethylenediamine triacetic acid is hydrolyzed, which is then bonded to the surface of the magnetic material particle. A nickel atom is chelate-bonded to the resulting magnetic material particle. This vertical link is regarded as one unit of a nickel chelate resin. As one unit on the right-hand side and one unit on the left-hand side are shown in Figure 1, silicon atoms Si of two units are connected laterally to each other through a -O-Si-O- bond to make a structure stronger, so that the silicon atom is hardly exfoliated from the surface of the magnetic material particle. The inexpensive nickel chelate resin has high adsorptivity of histidine. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a metal chelate compound that can be used for a carrier matrix or magnetic particles suitable for metal chelate / affinity chromatography, and a method for producing the same.

As a method for separating and purifying a protein, a plurality of histidines are connected to the end of the protein using a genetic recombination technique to obtain a so-called His Tag fusion protein.
When there is a metal ion such as Ni2 + or Co2 + which is bonded and fixed to the CH2COO group on the surface of the carrier matrix such as agarose or magnetic particles by chelate bond,
It interacts reversibly with the electron donating group located in the imidazole side chain of the plurality of histidines attached to the end of the protein.
That is, the His Tag fusion protein has a p-type in which the electron-donating group is partially unprotonated.
It binds to metal ions such as Ni2 + and Co2 + in H-value buffers, and then from metal ions such as Ni2 + and Co2 + in low-pH buffers where electron donating groups are protonated. Elute.
Previously used iminodiacetic acid has only two CH2COO groups, and therefore CH2COO.
When binding reversibly to the electron-donating group located in the imidazole side chain of the plurality of histidines, the binding force is weak for metal ions such as Ni2 + and Co2 + chelated to the group, Metal ions such as Ni 2+ and Co 2+ are easily separated from the CH 2 COO group, and are often washed out. (Patent Document 1)
However, Ni-NTA (nickel nitrilotriacetic acid) has three CH2COO groups, Ni2 +
Since the Co2 + ion has 6-coordination, 3 of them coordinate with 3 oxygens without the double bond of 3 CH2COO groups, and 1 coordinate with nitrogen. And each of the remaining two coordinations is 2
Coordinates with the imidazole side chain of a single histidine.
For this reason, metal ions such as Ni2 + and Co2 + have the advantage that they are difficult to separate from the CH2COO group and difficult to be washed out. However, when binding Ni-NTA (nickel nitrilotriacetic acid) to the carrier matrix or magnetic particles,
A compound called AB-NTA was devised, and this AB-NTA resin is extremely expensive. For example, the price of Dojindo Chemical's AB-NTA resin, one of the manufacturers, is as high as 110,000 yen per gram.
Using such expensive AB-NTA, for example, the surface of magnetic particles (diameter 150 nm)
In order to cover with AB-NTA resin, 300g of AB-NTA resin including wear must be used for 1Kg of magnetic particles, and the price is 300 x 110,000 yen = 33 million yen. .
In fact, a commercial product in which AB-NTA resin was bound to agarose spheres encapsulating magnetite was thus very expensive.

In metal chelate affinity chromatography, purification using magnetic particles is particularly important.
When affinity chromatography purification is performed by packing conventional non-magnetic agarose spheres (3 to 100 μm) into a liquid chromatographic column, first, so-called lysate containing proteins and various impurities is supplied from above the column. The His Tag fusion protein is specifically adsorbed in the space between the agarose spheres above, but at the same time, it is inevitable that various impurities are physically sandwiched in the space between the agarose spheres and adsorbed nonspecifically.
The lower the column, the less nonspecific adsorption of impurities, but the next time the His Tag fusion protein is washed out with a low pH buffer, it is unavoidable that these impurities will also be washed out. Absent.
For this reason, the purity of the His Tag fusion protein cannot be improved by passing through the column once, and it was necessary to pass through the column many times.
The service life of the agarose spheres was at most four times, and the column was disassembled every four times and the agarose spheres were discarded and refilled with new agarose spheres.
For this reason, continuous purification was impossible, and it was a batch method.

On the other hand, the purification method using magnetic particles uses His on the surface of magnetic particles dispersed in a buffer solution.
Since Tag fusion protein is specifically adsorbed, it is easy to stir the magnetic particles in the liquid by pipetting or the like. For this reason, the various impurities are not easily sandwiched between the particles and are easily eluted in the liquid. When the magnetic particles are adsorbed on the inner wall of the container using a magnet, impurities and proteins other than the His Tag fusion protein are eluted in the residual liquid, and the residual liquid can be easily removed. Next, if the His Tag fusion protein is washed out by running a buffer solution with a low pH value, high purity His
Since Tag fusion protein can be collected, the purification degree of His Tag fusion protein can be easily increased.
Moreover, since the purification rate is faster than that of the column and it is suitable for continuous purification, the magnetic particle method has been attracting attention as an effective method for mass purification from an early stage.

However, as described above, the price of the magnetic particles to which the metal chelate resin is bonded is too expensive, so that it could not be used for mass purification of the His Tag fusion protein even though it was known to be good.
If there is a metal chelate resin with three CH2COO groups that have strong binding force against Ni2 + and Co2 + ions, such as AB-NTA resin, if the price of magnetic particles bound with metal chelate resin is low, It is clear that the above problem is solved immediately and can be used for mass purification of His Tag fusion protein. The present invention is intended to solve the above problems.

The metal chelate compound that can be used for the carrier matrix or the magnetic particles is not directly connected to the carrier matrix or the magnetic particles, but the metal chelate compound is connected to the end of the spacer through a molecular chain called a spacer. My goal is,
In order to make the metal part of the metal chelate compound close to the histidine there even if the histidine part of the His Tag fusion protein is located in the back part of the three-dimensional structure of the protein by folding. Is used.
This spacer is essential. Although the metal chelate compound itself is inexpensive, the reason why the metal chelate compound to which the conventionally used spacer is connected is extremely expensive was found, and as a result, it was found that there should be a metal chelate compound having an inexpensive spacer. did. As a result of the efforts, we succeeded in developing a metal chelate compound with an inexpensive spacer.

As a result, an oxygen atom is bonded to a metal atom near the surface of the magnetic iron oxide, a first bond of a silicon atom is bonded to the oxygen atom, and (CH2) n is bonded to a second bond of the silicon atom. A carbon atom at one end of a chain hydrocarbon consisting of (n = 3 to 10) was bonded to form a spacer.
Then, the carbon atom at the other end of the chain hydrocarbon is bonded to the nitrogen atom of the ethylenediamine tricomplex acid that is bonded to one CH2COO group, to the three CH2COO groups of the ethylenediamine tricomplex acid. In which a divalent metal such as a nickel or cobalt atom is coordinated to form a unit metal chelate compound, and in order to connect a plurality of the unit metal chelate compounds, at least a third of the silicon atoms is connected. A silicon atom of another unit of a metal chelate compound having a similar structural formula is bonded to the bond through a bond represented by (—O—Si—) m—O— (m = integer). And the third bond of Si in the (—O—Si—) m—O— bond is bonded to the metal atom on the surface of the magnetic particle through oxygen,
We succeeded in producing a metal chelate compound in which a plurality of metal chelate compounds are bonded through silicon atoms, and have produced magnetite magnetic particles (50 nm or 400 nm) having the metal chelate compound in the plurality of bonds on the surface. And, as a result of purifying His Tag fusion protein by the known magnetic particle method that stirs magnetic particles in liquid by pipetting and collects magnetic particles using a magnet, We succeeded in developing magnetite magnetic particles with a metal chelate compound on the surface with high yield and very little adsorption of nonspecific impurities.
Moreover, it was confirmed that the material cost was extremely low at 800,000 yen per 1 kg of magnetite magnetic particles.

The cause is as follows.
AB-NTA (N- (5
Amino-1-carboxypentyl) iminodiacetic acid) itself requires a very expensive process. NTA (Nickel Nitrile Acetic Acid) has 3 -CH2COOH per nitrogen atom
Since it is a simple structure with a group attached, NH2— (CH2) 5— as a spacer can be bound to it by removing only one hydrogen atom from the CH2 part of one CH2COOH group and The rightmost carbon atom of NH2- (CH2) 5- must be connected. Therefore, it is unavoidable that it becomes expensive. If it is expensive, the amount of use will be small, and cost reduction has become increasingly difficult.

In order to solve this problem, a sodium salt of ethylenediaminetetraacetic acid (EDTA), which is extremely inexpensive and frequently used, was used. Then, it was found that it is possible to remove one -CH2COONa and then connect a trimethoxysilylpropyl group as a spacer in a relatively inexpensive process.
In fact, there is N- (trimethoxysilylpropyl) ethylenediaminetriacetic acid as an existing chemical substance, and a 50% aqueous solution of this Na salt is commercially available at a very low price of 18,000 yen at 25 g. In the present invention, this commercially available N- (trimethoxysilylpropyl) ethylenediamine triacetic acid was used. This material was modified in order to bind it to the magnetite surface efficiently and firmly.

First, 50% of sodium salt of N- (trimethoxysilylpropyl) ethylenediamine triacetate
Prepare 50 μl (50 M) of an aqueous solution, and add a small amount of tetraalkoxysilane such as tetramethoxysilane or tetraethoxysilane.
Add 3 ml of pure water, add a small amount of 35% aqueous ammonia, and stir with a magnetic stirrer. This beaker is referred to as a beaker 1.
Next, 0.1 g of magnetite particles is taken into another flask and 3 ml of pure water is added. 12 μl of the surfactant Tween 20 is added to it and stirred with a magnetic stirrer. Thereafter, an alternating magnetic field that attenuates is applied to demagnetize the residual magnetization of the magnetic particles. The purpose of degaussing is that when the magnet is applied, the magnetic particles will form clusters with each other if the magnetic material has residual magnetization.
This is because each particle is difficult to loosen. Next, ultrasonic waves are applied to disperse the magnetite.
The contents of the beaker 1 are added to it and a silanization reaction is performed with a shaker.
Thereafter, the magnetic particles are collected with a magnet, only the liquid part is removed, and the magnetite part is washed once with pure water. An alternating magnetic field that attenuates is applied to demagnetize the residual magnetization of the magnetic particles.

Thereafter, 20 ml of 10 mM nickel chloride and 5 mM glycine (PH = 8) aqueous solution are added. Shake for 3 hours on a shaker.
Thereafter, the magnetic particles are collected with a magnet, and only the liquid portion is removed, and the magnetite portion is washed once with pure water. An alternating magnetic field that decays is applied to demagnetize the residual magnetization of the magnetic particles. 1m to this
Add l pure water and transfer to a bottle. Thus, a dispersion of magnetite magnetic particles having a Ni chelate compound on the surface can be formed. The alternating magnetic field that attenuates is illustrated in “Patent Document 3” of the same applicant, and is not illustrated here, but is a magnetic field similar to the magnetic field inside the bulk eraser of the magnetic tape or the stator of the AC motor. Another embodiment of the damped alternating magnetic field may be an alternating magnetic field that rotates the magnet to create a rotating magnetic field and away from it. This is the details of the contents described in the first aspect. (Patent Document 3)

The structure of the Ni chelate compound bonded to the surface of magnetite in this dispersion is shown in FIG.
Shown in
In order to connect a plurality of metal chelate compounds of one unit, the third bond not bonded to the oxygen atom on the surface of the magnetite of the silicon atom in the metal chelate compound (-O-Si-)
A silicon atom of another unit of a metal chelate compound having a similar structural formula adjacent thereto was bonded through a bond represented by m-O- (m = integer). The metal chelate compound is a metal chelate compound in which a plurality of metal chelate compounds are bonded via a silicon atom, and in the (—O—Si—) m—O— bond.
The third bond of Si is a metal chelate compound characterized in that it is bonded to the metal atom on the surface of the magnetic particles through oxygen.
In addition, the tetra-alkoxysilane, ie, tetra-methoxysilane or tetra-
Ethoxysilane etc. can be hydrolyzed to form (—O—Si—) m—O— (m = integer) bond, and a plurality of the above-mentioned metal chelate compounds are bonded via silicon atoms and firmly attached to the magnetite surface. (—O—Si—) m as a so-called “lateral” bridge for bonding metal chelate compounds to
-O-bonds contribute, silicon in a plurality of adjacent metal chelate compounds are bonded laterally via (-O-Si-) m-O- bonds at the base, and ( -O-Si-) m-O
-A metal chelate compound characterized in that the third bond of Si in the bond is bonded to the metal atom on the surface of the magnetic particles through oxygen.
Since tetra-methoxysilane is used here, Si in the (-O-Si-) m-O- bond is used.
The fourth bond has a hydroxyl group.
It is also possible to use alkyl tri-alkoxysilane. In this case, the hydroxyl group (OH group) in FIG. 1 is replaced with an alkyl group.
FIG. 1 shows the contents described in the fourth aspect.
Here, the definition of “lateral direction” is as follows. When the direction in which the metal chelate compound spacer extends when the magnetic particle surface is viewed downward is defined as the “longitudinal direction”, silicon in a plurality of adjacent metal chelate compounds is at the base (—O—Si— ) The direction connected through the mO-bond is defined as "lateral direction".
In the magnetic particle surface B1, the B1 side shows the inside of the magnetic particles. Si represents a silicon atom on the surface of the magnetic particles. A silicon atom represented by Si is bonded to this silicon atom through oxygen O, and nickel atom represented by Ni is chelate-bonded to Si with propylethylenediamine triacetic acid extending upward. The name including Si is silylpropylethylenediamine triacetic acid. This vertical connection is defined as one unit of nickel chelate resin.
One unit on the left and one unit on the right are shown, but the silicon atom Si of each unit is (-O-Si-O-)
They are connected laterally to each other through a coupling. Although two units are shown here, the same applies to a plurality of units. The third bond of Si in (—O—Si—O—) is bonded to the silicon atom on the surface of the magnetic particles through oxygen.

However, FIG. 3 which is an embodiment using no alkyl trialkoxysilane, such as methyl trimethoxysilane or tetraalkoxysilane, is different from FIG.
In FIG. 3, the portion where nickel atom shown by Ni is chelate-bonded to propylethylenediamine triacetic acid is shown in a trident, but ethylenediamine triacetic acid is more spatially spread than NTA, so (−O— If they are not connected laterally to each other via Si—O—) bonds, the two ethylenediaminetriacetic acids are too close to each other and the distance between them is not appropriate.
However, in FIG. 1, the distance is appropriate, the required amount of propylethylenediamine triacetic acid is small, the cost is low, and the bonding to the silicon atoms on the surface of the magnetic particles is ensured.

Since tetraalkoxysilane is hydrolyzed, the trimethoxy silylpropyl portion of N- (trimethoxy silylpropyl) ethylenediamine triacetate works together with the trimethoxy hydrolysis of N- (trimethoxy silylpropyl) ethylenediamine triacetate. Hydrolyzed and bonded to metal atoms on the surface of magnetite or silicon atoms of SiO2 formed on the surface of magnetite, and then adjacent silicon atoms of silylpropyl are (-O-Si-) m-O.
-Bond and form cross-links in the transverse direction to form strong Ni chelates. The surface of magnetite may be directly exposed with iron atoms or covered with a SiO2 film.

Also, Ni chelate resin with three CH2COO groups, reinforced with lateral cross-linking, is His
Tag
It is particularly suitable for mass purification of fusion proteins. When mass purification of His Tag fusion protein is performed by the magnetic particle method, the magnetic particles are vigorously stirred to a certain extent, so the magnetic particles are firmly protected so that the Ni chelate resin on the surface of the magnetic particles does not peel off from the surface of the magnetic particles. It is necessary to adhere to the surface, and from this point as well, lateral cross-linking by (—O—Si—) m—O— bond is performed at the base,
Inexpensive and high performance magnetic particles having a Ni chelate resin strongly bonded to the surface of the magnetic particles are suitable for this purpose.
In addition, Ni chelate resin with three CH2COO groups can be provided without using expensive AB-NTA resin, thus opening the way to low-cost and rapid collection and purification of proteins, which is an important issue for post-genome. become.

An oxygen atom is bonded to a metal atom near the surface of the magnetic iron oxide, a first bond of a silicon atom is bonded to the oxygen atom, and a second bond of the silicon atom is composed of (CH2) n. A carbon atom at one end of a chain hydrocarbon (n = 3 to 10) is bonded, and a carbon atom at the other end of the chain hydrocarbon is bonded to one CH2COO group of nitrogen atoms of ethylenediamine tricomplex. In which a divalent metal such as a nickel or cobalt atom is coordinated to the three CH2COO groups of ethylenediamine tricomplex acid to form one unit of a metal chelate compound. In order to connect a plurality of Ni chelate compounds, at least a third bond of the silicon atom is adjacent to each other through a bond represented by (—O—Si—) m—O— (m = integer). Of another unit of Ni chelate with the structural formula The third bond of Si in the (—O—Si—) m—O— bond is bonded to the metal atom on the surface of the magnetic particle through oxygen. The best mode is to produce a metal chelate compound in which a plurality of silicon atoms are bonded through silicon atoms.
Here, the value of m of the (—O—Si—) m—O— bond is an integer of 0 or more. Of course, even when m = 0 as shown in FIG.

The present invention opens the way to high-purity, low-cost, high-speed, continuous mass collection and purification of post-genomic proteins. By eluting His Tag fusion protein from magnetic particles, His
A large amount of Tag fusion protein can be collected and purified. The His Tag part can be removed using an enzyme.
It can be a protein without a tag.
In addition, a drug discovery screening system can be constructed. When magnetic particles with the His Tag fusion protein fixed are dispersed, for example, in the ascites of a living body, antibodies that have an affinity for the His Tag fusion protein, proteins with particular affinity for the His Tag fusion protein, DNA, etc. , His
Because it specifically adsorbs to the Tag fusion protein, the magnetic particles are collected using a magnet, and the antibodies,
Purification can be achieved by eluting protein, DNA and the like.
Conventionally, antibody purification using magnetic particles has been performed on a trial basis, but the magnetic particles are too expensive to be used for mass purification.
However, according to the present invention, it is possible to provide inexpensive magnetic particles.
It is possible to collect and purify a large amount of antibodies and DNA having affinity especially for antibodies using Tag fusion proteins as antigens, His Tag fusion proteins, and DNA.
The magnetic iron oxide may be a metal chelate resin characterized in that the surface thereof is magnetite, nickel zinc ferrite, or manganese zinc ferrite with or without silica coating. It is clear that N- (trimethoxy silylpropyl) ethylenediamine triacetic acid can be hydrolyzed to connect with —O—Si— to the metal atoms on the surface.
The ethylenediamine tricomplex acid referred to in the present invention means that N at the left end of N-CH2-CH2-N is 2
It is ethylenediamine tricomplex acid in which one CH2COO group is bonded, and one CH2COO group is bonded to N at the right end.

First, 50% of sodium salt of N- (trimethoxysilylpropyl) ethylenediamine triacetate
Prepare 50 μl (50 M) of an aqueous solution, and add a small amount of tetraethoxysilane.
To this, 3 ml of pure water is added, then a small amount of 35% ammonia water is added, and the mixture is stirred with a magnetic stirrer. This beaker is referred to as a beaker 1.
Next, 0.1 g of magnetite particles is taken into another flask and 3 ml of pure water is added. 12 μl of the surfactant Tween 20 is added to it and stirred with a magnetic stirrer. Thereafter, an alternating magnetic field that attenuates is applied to demagnetize the residual magnetization of the magnetic particles. The purpose of degaussing is that when the magnet is applied, the magnetic particles will form clusters with each other if the magnetic material has residual magnetization.
This is because each particle is difficult to loosen. Next, ultrasonic waves are applied to disperse the magnetite.
The contents of the beaker 1 are added to it and a silanization reaction is carried out with a shaker.
Thereafter, the magnetic particles are collected with a magnet, only the liquid part is removed, and the magnetite part is washed once with pure water. An alternating magnetic field that decays is applied to demagnetize the residual magnetization of the magnetic particles.

Thereafter, 20 ml of 10 mM nickel chloride and 5 mM glycine (PH = 8) aqueous solution are added. Shake for 3 hours on a shaker.
Thereafter, the magnetic particles are collected with a magnet, and only the liquid portion is removed, and the magnetite portion is washed once with pure water. An alternating magnetic field that decays is applied to demagnetize the residual magnetization of the magnetic particles. 1ml of this
Add pure water and transfer to a bottle. Thus, a dispersion of magnetite magnetic particles having a Ni chelate compound on the surface can be formed.

As an example, when β-galactosidase is expressed using Escherichia coli, a His Tag fusion protein with 6 histidines attached to the C-terminus is expressed using a genetic recombination method.
Next, Escherichia coli is sonicated and the supernatant is taken, and this is used as the original lysate.
Take 200 μl of this lysate in a 1 ml Eppendorf tube, and add magnetic particle dispersion 2
Add 00 μL, stir by pipetting, mix the solution, and incubate at room temperature for 2 minutes.
A magnet is applied to the side wall of the tube for 30 seconds, and the solution portion is removed with a pipette while the magnetic particles are captured by the magnet. Then move the magnet away.
Next, Wash Buffer 1 containing 20 mM imidazole is added and stirred by pipetting. A magnet is applied to the side wall of the tube for 30 seconds, and the solution portion is removed with a pipette while the magnetic particles are captured by the magnet. Then move the magnet away.
Next, add Wash Buffer 2 containing 20 mM imidazole and stir by pipetting.
A magnet is applied to the side wall of the tube for 30 seconds, and the solution portion is removed with a pipette while the magnetic particles are captured by the magnet. Then move the magnet away.
Add Wash Buffer 3 containing 40 mM imidazole and stir by pipetting.
A magnet is applied to the side wall of the tube for 30 seconds, and the solution portion is removed with a pipette while the magnetic particles are captured by the magnet. Then move the magnet away.
Finally, add Elution Buffer containing 300 mM imidazole and stir by pipetting.
When a magnet is applied to the side wall of the tube for 30 seconds and the magnetic particles are trapped by the magnet, the solution part is sucked with a pipette and separated into another tube.
Tag fusion protein is collected (E1 in FIG. 2). Elution was performed once more (E2 in FIG. 2).

The collected His Tag fusion protein is subjected to SDS PAGE (polyacrylamide gel electrophoresis).

The result is shown in FIG.
The band shown on the left in the figure is purified using expensive Ni agarose magnetite magnetic particles from other companies, indicating that the band is thin and the yield is low.
The band shown on the right side of the figure is purified using the magnetic particles of the present invention.
The darkness of the band indicates that the yield is high and the yield is high. E1 is the first Elution fraction, E2
Indicates the second Elution fraction. The band shown on the right in the figure is still His even in E2.
Tag
The fusion protein is Elution, and this also indicates that the ability to collect the His Tag fusion protein is large.
Usually, as the band of His Tag fusion protein becomes darker, the non-specific adsorption band also becomes thicker. However, the band shown on the right in the figure has a higher band of His Tag fusion protein. Nevertheless, nonspecific adsorption bands are not visible.

This experiment shows that, when the His Tag fusion protein is collected and purified with the Ni chelate resin of the present invention, the 6 histidine tags present at the end of the protein can be adsorbed very selectively.
Moreover, the difficulty of the collection ability of the His Tag fusion protein which is a defect of N, N, N-ethylenediaminetriacetic acid shown in Non-Patent Document 1 mentioned in Patent Document 2 is that the Ni chelate resin of the present invention is not suitable. It is shown that the problem is solved with the magnetic particles.
Porah, Nature258,598-599, (1975) JP-A-8-99944 Haner.M, Anal.Biochem, 138, 229-234 (1984) Japanese Patent Application No. 2003-367637 “Separation of biological constituents”

In the magnetic particle surface B1, the B1 side shows the inside of the magnetic particles. Si represents a silicon atom on the surface of the magnetic particles. A silicon atom represented by Si is bonded to this silicon atom through oxygen O, and nickel atom represented by Ni is chelate-bonded to Si with propylethylenediamine triacetic acid extending upward. The name including Si is silylpropylethylenediamine triacetic acid. This vertical connection is defined as one unit of nickel chelate resin. Although one unit on the left and one unit on the right are shown, the silicon atoms Si of each unit are connected to each other laterally via a (—O—Si—O—) bond. Although two units are shown here, the same applies to a plurality of units. The third bond of Si in (—O—Si—O—) is bonded to the silicon atom on the surface of the magnetic particles through oxygen. The band shown on the left in the figure is purified using expensive magnetic particles from a foreign company, and the band is thin and the yield is low. The band shown on the right side of the figure is purified using the magnetic particles of the present invention, and the band is dark and has a high yield. E1 represents the first Elution, and E2 represents the second Elution. The band shown on the right in the figure shows that the His Tag fusion protein is still Elution even in E2, and this also shows that the collection ability of the His Tag fusion protein is large. In the magnetic particle surface B1, the B1 side shows the inside of the magnetic particles. Si represents a silicon atom on the surface of the magnetic particles. A silicon atom represented by Si is bonded to this silicon atom through oxygen O, and nickel atom represented by Ni is chelate-bonded to Si with propylethylenediamine triacetic acid extending upward. The name including Si is silylpropylethylenediamine triacetic acid. This vertical connection is defined as one unit of nickel chelate resin. The portion where nickel atom shown by Ni is chelate-bonded to propylethylenediamine triacetic acid is shown in a trident, but ethylenediamine triacetic acid is more spatially spread than NTA, so (-O-Si-O as shown in Fig. 1). -) If they are not connected laterally to each other via a bond, the two ethylenediaminetriacetic acids are too close to each other and the distance between them is not appropriate.

Explanation of symbols

B1; indicates the surface of the magnetic particles, and the B1 side indicates the inside of the magnetic particles.
Ni: Nickel atom.
Si: Silicon atom.
O: an oxygen atom.
OH: hydroxyl group.
Zigzag line and trident line extending above Si; propylethylenediamine triacetic acid is shown.

Claims (4)

An oxygen atom is bonded to a metal atom near the surface of the magnetic iron oxide, a first bond of a silicon atom is bonded to the oxygen atom, and a second bond of the silicon atom is composed of (CH2) n. A carbon atom at one end of a chain hydrocarbon (n = 3 to 10) is bonded, and a carbon atom at the other end of the chain hydrocarbon is bonded to one CH2COO group of nitrogen atoms of ethylenediamine tricomplex. In which a divalent metal such as a nickel or cobalt atom is coordinated to the three CH2COO groups of ethylenediamine tricomplex acid to form one unit of a metal chelate compound. In order to connect a plurality of metal chelate compounds, at least a third bond of the silicon atom is adjacent to each other via a bond represented by (—O—Si—) m—O— (m = integer). Another unit chelate compound with a complex structure When manufacturing a metal chelate compound in which a plurality of the unit metal chelate compounds are bonded, the magnetic iron oxide is dispersed in a liquid using ultrasonic waves. Then, after collecting the magnetic iron oxide using a magnet, the liquid part is removed, and then the residual magnetization of the magnetic iron oxide is demagnetized using an alternating magnetic field that attenuates, and another ultrasonic wave is used to demagnetize the remaining iron oxide. A dispersion of a metal chelate compound in which a plurality of the unit metal chelate compounds are bonded to the surface of magnetic particles, wherein the metal chelate compound is dispersed in a liquid. An oxygen atom is bonded to a metal atom near the surface of the magnetic iron oxide, a first bond of a silicon atom is bonded to the oxygen atom, and a second bond of the silicon atom is composed of (CH2) n. A carbon atom at one end of a chain hydrocarbon (n = 3 to 10) is bonded, and a carbon atom at the other end of the chain hydrocarbon is bonded to one CH2COO group of nitrogen atoms of ethylenediamine tricomplex. In which a divalent metal such as a nickel or cobalt atom is coordinated to the three CH2COO groups of ethylenediamine tricomplex acid to form one unit of a metal chelate compound. In order to connect a plurality of metal chelate compounds, at least a third bond of the silicon atom is adjacent to each other via a bond represented by (—O—Si—) m—O— (m = integer). Another unit chelate compound with a complex structure A dispersion of a metal chelate compound in which a plurality of the single unit metal chelate compounds are bound, wherein silicon atoms of the product are bound. The third bond of Si in the (—O—Si—) m—O— bond is bonded to a metal atom on the surface of the magnetic particle through oxygen. To
A dispersion of a metal chelate compound in which a plurality of metal chelate compounds bonded through silicon atoms are bonded to the surface of magnetic particles. The silicon atom according to claim 1 or 2, wherein a hydroxyl group (OH group) is connected to the fourth bond of Si in the (-O-Si-) mO- bond. A dispersion of a metal chelate compound in which a plurality of metal chelate compounds bonded to each other are bonded to the surface of magnetic particles.

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