U.S. patent application number 13/740763 was filed with the patent office on 2013-06-27 for carrier polymer particle, process for producing the same, magnetic particle for specific trapping, and process for producing the same.
The applicant listed for this patent is Tetsuo Fukuda, Kiyoshi Kasai, Satoshi Katayose, Toshihiro Ogawa, Masayuki TAKAHASHI, Kouji Tamori, Kinji Yamada. Invention is credited to Tetsuo Fukuda, Kiyoshi Kasai, Satoshi Katayose, Toshihiro Ogawa, Masayuki TAKAHASHI, Kouji Tamori, Kinji Yamada.
Application Number | 20130164761 13/740763 |
Document ID | / |
Family ID | 37431259 |
Filed Date | 2013-06-27 |
United States Patent
Application |
20130164761 |
Kind Code |
A1 |
TAKAHASHI; Masayuki ; et
al. |
June 27, 2013 |
CARRIER POLYMER PARTICLE, PROCESS FOR PRODUCING THE SAME, MAGNETIC
PARTICLE FOR SPECIFIC TRAPPING, AND PROCESS FOR PRODUCING THE
SAME
Abstract
Carrier polymer particles comprising organic polymer particles
having a particle diameter of 0.1 to 20 micrometers and a
saccharide with which the surface of the organic polymer particles
is covered, the organic polymer particles and the saccharide being
chemically bonded.
Inventors: |
TAKAHASHI; Masayuki;
(Tsukuba-shi, JP) ; Fukuda; Tetsuo;
(Tsuchiura-shi, JP) ; Kasai; Kiyoshi;
(Kawasaki-shi, JP) ; Ogawa; Toshihiro;
(Tsukuba-shi, JP) ; Katayose; Satoshi;
(Tsukubamirai-shi, JP) ; Tamori; Kouji;
(Tsuchiura-shi, JP) ; Yamada; Kinji; (Tsukuba-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TAKAHASHI; Masayuki
Fukuda; Tetsuo
Kasai; Kiyoshi
Ogawa; Toshihiro
Katayose; Satoshi
Tamori; Kouji
Yamada; Kinji |
Tsukuba-shi
Tsuchiura-shi
Kawasaki-shi
Tsukuba-shi
Tsukubamirai-shi
Tsuchiura-shi
Tsukuba-shi |
|
JP
JP
JP
JP
JP
JP
JP |
|
|
Family ID: |
37431259 |
Appl. No.: |
13/740763 |
Filed: |
January 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13025273 |
Feb 11, 2011 |
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13740763 |
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11914986 |
Mar 3, 2008 |
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PCT/JP2006/309810 |
May 17, 2006 |
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13025273 |
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Current U.S.
Class: |
435/7.1 ;
436/501 |
Current CPC
Class: |
G01N 33/53 20130101;
B32B 5/16 20130101; C08G 63/91 20130101; G01N 33/5434 20130101;
Y10T 428/2998 20150115; Y10T 428/2982 20150115; G01N 33/54353
20130101 |
Class at
Publication: |
435/7.1 ;
436/501 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2005 |
JP |
2005-147824 |
Sep 22, 2005 |
JP |
2005-276052 |
Nov 28, 2005 |
JP |
2005-342167 |
Claims
1. A process for producing a magnetic particle composition
comprising: chemically bonding at least one magnetic particle
having a diameter of 0.1 to 20 micrometers to a saccharide and
chemically bonding a probe for specifically trapping a target
material to the saccharide, wherein the probe is bonded to the
saccharide of the magnetic particle after treatment of the magnetic
particle with a basic solution.
2. The process according to claim 1, wherein when chemically
bonding the magnetic particles and the saccharide, the at least one
magnetic particle has a first functional group and the saccharide
has a second functional group, and the at least one magnetic
particle and the saccharide are chemically bonded by reacting the
first functional group and the second functional group.
3. The process according to claim 2, wherein the first functional
group is at least one functional group selected from the group
consisting of a carboxyl group, an epoxy group, an amino group, and
a tosyl group.
4. The process according to claim 1, wherein the saccharide is a
polysaccharide.
5. The process according to claim 1, wherein the saccharide is
carboxymethylated.
6. The process according to claim 1, wherein the magnetic particle
and the saccharide are chemically bonded by a bonding group which
comprises at least one selected from the group consisting of an
amide bond and an ester bond.
7. The process according to claim 1, wherein the at least one
magnetic particle is obtained by a process comprising polymerizing
a polymer layer on a magnetic material layer comprising a mother
particle, the mother particle comprising at least one nuclear
particle and the magnetic material layer formed on the surface of
the nuclear particle, and wherein the magnetic material layer
comprises at least one of Fe.sub.2O.sub.3 and Fe.sub.3O.sub.4.
8. The process according to claim 1, wherein the probe is at least
one probe selected from the group consisting of a protein, a
peptide, a nucleic acid, a glycoside compound, and a synthetic
chemical material.
9. The process according to claim 1, wherein the saccharide and the
probe are bonded by a chemical bond selected from the group
consisting of an --O-- bond, an --S-- bond, an --SO-- bond, an
--SO.sub.2-- bond, a --CO-- bond, a --CO.sub.2-- bond, an --NR--
bond (wherein R is an alkyl group or H), an
--N.sup.+R.sup.2R.sup.3-- bond (wherein R.sup.2 and R.sup.3 are
individually an alkyl group or H), an --NHCO-- bond, and a
--PO.sub.2-- bond.
10. The process according to claim 1, wherein the saccharide and
the probe are bonded by a process comprising chemically reacting a
functional group in the saccharide with a functional group in the
probe.
11. The process according to claim 1, wherein the functional group
in the saccharide and the functional group in the probe are at
least one selected from the group consisting of a hydroxyl group,
an acyl group, a mercapto group, an amino group, an amino acyl
group, a carbonyl group, a formyl group, a carboxyl group, an amide
group, a sulfonic group, a phosphate group, an epoxy group, a tosyl
group, an azido group, a vinyl group, and an allyl group.
12. The process according to claim 1, wherein the target material
is a biological material.
13. The process according to claim 1, wherein the biological
material is at least one selected from the group consisting of a
protein, a peptide, a nucleic acid, a carbohydrate, a lipid, or
another cellular substance.
14. The process according to claim 1, wherein the other cellular
substance is a blood-originating substance, a floating cell, a
platelet, a erythrocyte, a leukocytes, or mixtures thereof.
15. The process according to claim 1, wherein said particle is
dispersed in a dispersion medium.
16. A process of specific trapping comprising trapping a target
material in the presence of the magnetic particle formed by the
process of claim 1.
Description
[0001] This application is a divisional application of U.S. Ser.
No. 13/025,273, which is a divisional application of U.S.
application Ser. No. 11/914,986, filed on Mar. 3, 2008, which is a
371 of PCT/JP06/309810, filed on May 17, 2006, and claims priority
to Japanese Patent Application No. 2005-342167, filed on Nov. 28,
2005, Japanese Patent Application No. 2005-276052, filed on Sep.
22, 2005, and Japanese Patent Application No. 2005-147824, filed on
Mar. 20, 2005.
TECHNICAL FIELD
[0002] The present invention relates to carrier polymer particles
in which the surface of organic polymer particles are covered with
a saccharide, a process for producing the same, magnetic particles
for specific trapping, and a process for producing the same.
BACKGROUND ART
[0003] In recent years, attempts have been actively made in fields
such as drug discovery to find molecules having specific
interaction with a certain specific molecule by utilizing
intermolecular interaction. Specifically, immobilizing a molecule
(probe molecule) having interaction on a support, and trapping and
purifying another molecule (target material) by utilizing a
specific interaction is widely carried out.
[0004] For example, the discovery of the intracellular binding
protein FKBP12 of the immunosuppressant FK506 using an affinity
resin (Nature, 341, 758, 1989) has been known. A porous gel such as
agarose is commonly used as such affinity resin. However, when
using a porous gel, the so-called phenomenon nonspecific adsorption
in which molecules other than the target molecule are adsorbed on
the affinity resin arises and thus, the problem that separation and
purification of the target molecule is difficult arises. A certain
proportion among the probe molecules bond internally to the porous
gel and as a result of such probe molecules having insufficient
interaction with the target material, the problem arises that
trapping efficiency of the target material is reduced.
[0005] As a solution to such nonspecific adsorption, microspheres
made from a styrene/glycidyl methacrylate polymer, of which the
surface is covered with glycidyl methacrylate, and a
biologically-related material bonded to the polymer through a
spacer have been proposed (JP-B-3086427 and JP-B-3292721). Also
disclosed are particles having a hydrophilic spacer introduced on
the surface (WO 2004/025297 A1 and WO 2004/040305A1), and the like.
However, none of these have a sufficient effect in lowering
nonspecific adsorption. Support particles having still smaller
nonspecific adsorption are desired. Also, the efficiency of
trapping the target material of these particles is not
sufficient.
[0006] On the other hand, as biologically-related material carrier
polymer particles which are sensitized by a chemical bonding
method, carboxyl group-modified polystyrene particles are widely
used. However, since the polystyrene particles generally have
significant capability of adsorbing other biologically-related
materials (nonspecific adsorption) which are not target materials
existing in the test sample, the performance of the sensitized
particles is inhibited, posing a serious obstacle to use of the
particles. In contrast, a blocking method, in which the surface of
the particles is first sensitized with the target
biologically-active material and a protein having little damage
such as bovine serum albumin (BSA) is adsorbed on the remaining
particle surface, has difficulty in fully preventing nonspecific
adsorption. Also, although it is known that performance of
polystyrene particles as biologically-related material carrier
particles can be improved by copolymerizing a styrene sulfonate or
an acrylic ester having a polyalkylene oxide side chain represented
by the formula (CH.sub.2CH.sub.2O).sub.n or
(CH.sub.2CHCH.sub.3O).sub.m or by hydrolyzing fragments of
persulfate initiator bonded to the particles by heat treatment in
an alkaline aqueous solution after emulsion polymerization of the
particles, the nonspecific adsorption is not sufficiently
prevented. Also, the efficiency of trapping the target material of
these particles is not sufficient.
DISCLOSURE OF THE INVENTION
[0007] An object of the invention is to provide carrier polymer
particles which have very small nonspecific absorption of
biological materials such as proteins and a process for producing
the same.
[0008] Another object of the invention is to provide carrier
polymer particles which have very small nonspecific absorption of
biologically-related materials such as proteins and which have a
high trapping efficiency of the target material, and a process for
producing the same.
[0009] A further object of the invention is to provide magnetic
particles for specific trapping which have very small nonspecific
absorption of biologically-related materials such as proteins,
peptides, nucleic acids, and cells and a process for producing the
same.
[0010] Carrier polymer particles according to a first aspect of the
invention comprise organic polymer particles having a particle
diameter of 0.1 to 20 micrometers and a saccharide with which the
surface of the organic polymer particles is covered, wherein the
organic polymer particles and the saccharide are chemically
bonded.
[0011] In the carrier polymer particles, the saccharide may be a
polysaccharide.
[0012] In the carrier polymer particles, the saccharide may be
carboxymethylated.
[0013] In the carrier polymer particles, the organic polymer
particles and the saccharide may be chemically bonded by a bonding
group including at least one of an amide bond and an ester
bond.
[0014] A process for producing carrier polymer particles according
to a second aspect of the invention comprises covering the surface
of organic polymer particles having a particle diameter of 0.1 to
20 micrometers with a saccharide by chemically bonding the organic
polymers and the saccharide.
[0015] When chemically bonding in the process for producing carrier
polymer particles, the organic polymer particles have a first
functional group and the saccharide has a second functional group,
and the organic polymer particles and the saccharide may be
chemically bonded by reacting the first functional group and the
second functional group.
[0016] In the process for producing carrier polymer particles, the
first functional group may be at least one functional group
selected from the group consisting of a carboxyl group, an epoxy
group, an amino group, and a tosyl group.
[0017] A process for producing carrier polymer particles according
to a third aspect of the invention comprises: [0018] covering
organic polymer particles having a particle diameter of 0.1 to 20
micrometers and a functional group having reactivity with a
carboxyl group with a saccharide having a carboxyl group by
chemically bonding the organic polymer particles and the
saccharide; and [0019] treating the organic polymer particles of
which the surface has been covered with the saccharide with a basic
solution.
[0020] In the process for producing carrier polymer particles, the
saccharide may be a polysaccharide.
[0021] In the process for producing the carrier polymer particles,
the chemically bonding may be achieved by a bonding group including
at least one of an amide bond and an ester bond.
[0022] In the process for producing carrier polymer particles, the
functional group having reactivity with the carboxyl group may be
at least one functional group selected from the group consisting of
an amino group, a hydroxyl group, and an epoxy group.
[0023] The process for producing the carrier polymer particles may
further comprise chemically bonding a probe for specifically
trapping a target material to the saccharide.
[0024] Magnetic particles for specific trapping according to a
fourth aspect of the invention comprise magnetic particles having a
particle diameter of 0.1 to 20 micrometers and a saccharide,
wherein the magnetic particles and the saccharide are chemically
bonded and a probe for specifically trapping a target material is
bonded to the saccharide.
[0025] The saccharide of the magnetic particles for specific
trapping may be a polysaccharide.
[0026] The saccharide of the magnetic particles for specific
trapping may be carboxymethylated.
[0027] The magnetic particles for specific trapping and the
saccharide may be chemically bonded by a bonding group including at
least one of an amide bond and an ester bond.
[0028] The magnetic particles of the magnetic particles for
specific trapping are obtained by polymerization of a polymer layer
on the magnetic material layer of mother particles comprising
nuclear particles and a magnetic material layer formed on the
surface of the nuclear particles, and the magnetic material layer
may include at least one of Fe.sub.2O.sub.3 and
Fe.sub.3O.sub.4.
[0029] The probe of the magnetic particles for specific trapping
may be at least one selected from proteins, peptides, nucleic
acids, glycoside compounds, and synthetic chemical materials.
[0030] A process for producing magnetic particles for specific
trapping according to a fifth aspect of the invention comprises:
[0031] chemically bonding magnetic particles having a particle
diameter of 0.1 to 20 micrometers and a saccharide; and [0032]
chemically bonding a probe for specifically trapping a target
material to the saccharide.
[0033] When chemically bonding the magnetic particles and the
saccharide in the process for producing carrier polymer particles,
the magnetic particles have a first functional group and the
saccharide has a second functional group, and the magnetic
particles and the saccharide may be chemically bonded by reacting
the first functional group and the second functional group.
[0034] In the process for producing magnetic particles for specific
trapping, the first functional group may be at least one functional
group selected from the group consisting of a carboxyl group, an
epoxy group, an amino group, and a tosyl group.
[0035] The carrier polymer particles possess the characteristic of
having little nonspecific adsorption due to the organic polymer
particles having a particle diameter of 0.1 to 20 micrometers, the
saccharide covering the surface of the organic polymer particles,
and the organic polymer and the saccharide are chemically bonded.
Thus, the separation and the purification of target molecules can
be easily carried out.
[0036] According to the process for producing carrier polymer
particles, carrier polymer particles having little nonspecific
adsorption and a high efficiency of trapping a target material can
be obtained by covering the organic polymer particles with a
particle diameter of 0.1 to 20 micrometers, which has a functional
group having reactivity with a carboxyl group, with a saccharide
having a carboxyl group by chemically bonding the organic polymer
particles and the saccharide, and treating the organic polymer
particles, of which the surface has been covered with the
saccharide, with a basic solution. Thus, the separation and the
purification of target molecules can be easily carried out.
[0037] Furthermore, the magnetic particles for specific trapping
possess the characteristic of having little nonspecific adsorption,
due to the use of the magnetic particles having a particle diameter
of 0.1 to 20 micrometers, inclusion of a saccharide, the magnetic
particles are chemically bonded to the saccharide, and a probe for
specifically trapping a target material is chemically bonded to the
saccharide. Thus, the separation and the purification of target
molecules can be easily carried out.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a diagram showing an example of a process for
producing carrier polymer particles according to the first and
second embodiments of the invention.
[0039] FIG. 2 is a diagram showing an example of carrier polymer
particles according to the first and second embodiments of the
invention.
[0040] FIG. 3 is a photograph showing the specific trapping
evaluation results of the probe-bonded particles obtained in
Experimental Examples 8 and 9, and Comparative Example 4
(electrophoresis pattern of the proteins adsorbed on the probe
bonding particles).
[0041] FIG. 4 is a photograph showing the specific trapping
evaluation results of the probe-bonded particles obtained in
Experimental Examples 10 and 11, and Comparative Example 7
(electrophoresis pattern of the proteins adsorbed on the probe
bonding particles).
BEST MODE FOR CARRYING OUT THE INVENTION
[0042] The carrier polymer particles, the process for producing the
same, the magnetic particles for specific trapping, and the process
for producing the same of the invention are explained in detail
below.
1. First Embodiment
1-1. Carrier Polymer Particles
[0043] The carrier polymer particles according to the first
embodiment of the invention comprise organic polymer particles
having a particle diameter of 0.1 to 20 micrometers and a
saccharide with which the surface of the organic polymer particles
is covered. Also, the organic polymer particles and the saccharide
are chemically bonded in the carrier polymer particles according to
this embodiment. Although not limited, it is preferable that the
chemically bonding is achieved by a bonding group including at
least one of an amide bond and an ester bond.
[0044] Although it is possible to use the carrier polymer particles
according to the present embodiment as they are, they can also be
used as a dispersion liquid in which the particles are dispersed in
a dispersion medium in order to efficiently carry out a reaction
with a compound. As examples of the dispersion medium, water;
alcohols such as methanol, ethanol, propanol, isopropyl alcohol,
n-butyl alcohol, isobutyl alcohol, sec-butyl alcohol, and
tert-butyl alcohol; ethylene glycol derivatives such as ethylene
glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl
ether, ethylene glycol monopropyl ether, ethylene glycol monobutyl
ether, ethylene glycol monoethyl ether acetate, diethylene glycol
monomethyl ether, diethylene glycol monoethyl ether, diethylene
glycol dimethyl ether, and diethylene glycol diethyl ether;
propylene glycol derivatives such as propylene glycol, propylene
glycol monomethyl ether, propylene glycol monoethyl ether,
propylene glycol monopropyl ether, propylene glycol monobutyl
ether, and propylene glycol monomethyl ether acetate; ketones such
as acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl
amyl ketone, diisobutyl ketone, and cyclohexanone; esters such as
ethyl acetate, butyl acetate, isobutyl acetate, ethyl lactate, and
gamma-butyl lactone; amides such as N,N-dimethylformamide,
N,N-dimethylacetamide, and N-methylpyrrolidone; dimethyl sulfoxide;
and aromatic hydrocarbons such as toluene and xylene can be
given.
[0045] The particle diameter of the carrier polymer particles
according to the present embodiment are preferably 0.1 to 17
micrometers, and more preferably 1 to 10 micrometers. When the
particle diameter is less than 0.1 micrometer, since separation
using centrifugal separation or the like takes a long time and
separation of the washing solvent such as water and the particles
is insufficient, there are situations in which the removal of
non-target molecules (for example, biologically-related materials
such as proteins) is insufficient and thus, sufficient purification
is not possible. In contrast, since particles with a diameter
exceeding 17 micrometers reduces the surface area of the particles,
there may be situations in which the trapped amount of the
biologically-related material such as proteins, which is the
target, is small.
[0046] Next, the constituting elements of the carrier polymer
particles according to the embodiment are explained in detail.
1-1-1. Organic Polymer Particles
[0047] The average particle diameter of the organic polymer
particles used in the present embodiment is 0.1 to 20 micrometers,
more preferably 0.3 to 15 micrometers, and most preferably 1 to 10
micrometers. Also, the coefficient of variation of the organic
polymer particles used in the invention is normally 30% or less,
preferably 20% or less, and more preferably 10% or less.
[0048] In the embodiment, the organic polymer particles may be used
as base particles of the carrier polymer particles according to the
embodiment. Organic polymer particles are suitable as base
particles, since it is easy to cover the surface of organic polymer
particles with a saccharide which is bonded by chemically bonding.
Also, magnetic particles may be used as the organic polymer
particles.
[0049] As explained above, when the carrier polymer particles
according to the embodiment are dispersed in a solvent, nonspecific
adsorption of biologically-related materials such as proteins
increases if the organic polymer particles are dispersed in a
dispersion medium or the organic polymer particles swell by the
solvent. For this reason, it is desirable that the organic polymer
particles do not dissolve in the dispersion medium. Here, an
aqueous medium may be used as the dispersion medium, for example.
Here, aqueous medium means water or a mixture of water and a
solvent which are dissolved in water (for example, alcohols and
alkylene glycol derivatives).
[0050] Vinyl polymers are particularly preferable as the polymer
constituting the organic polymer particles. As examples of the
vinyl monomers constituting the vinyl polymer, aromatic vinyl
monomers such as styrene, alpha-methyl styrene, halogenated
styrene, and divinylbenzene; vinyl esters such as vinyl acetate and
vinyl propionate; unsaturated nitriles such as acrylonitrile;
ethylenic unsaturated carboxylic acid alkyl esters such as methyl
acrylate, ethyl acrylate, ethyl methacrylate, butyl acrylate, butyl
methacrylate, 2-ethylhexyl acrylate, 2-ethylhexyl methacrylate,
lauryl acrylate, lauryl methacrylate, cyclohexyl acrylate, and
cyclohexyl methacrylate; polyfunctional (meth)acrylates such as
ethylene glycol diacrylate, ethylene glycol dimethacrylate,
trimethylol propane triacrylate, and trimethylol propane
trimethacrylate; and (meth)acrylates having a functional group such
as glycidyl acrylate, glycidyl methacrylate, 2-hydroxyethyl
acrylate, and 2-hydroxyethyl methacrylate can be given. The vinyl
polymer may be a homopolymer or may be a copolymer comprising two
or more monomers selected from the above-mentioned vinyl monomers.
Also, a copolymer of the above-mentioned vinyl monomers and
copolymerizable monomers such as conjugated diolefins such as
butadiene and isoprene, acrylic acid, methacrylic acid, itaconic
acid, acrylamide, methacrylamide, N-methylol acrylamide, N-methylol
methacrylamide, diallyl phthalate, allyl acrylate, and allyl
methacrylate can also be used.
[0051] The magnetic particles are general particulate materials
which can be magnetically collected and include fine particles of a
magnetic material. When the organic polymer particles used in this
embodiment are magnetic particles, the carrier polymer particles
according to the embodiment can be used as magnetic particles
usable in applications described later as examples.
[0052] If the particle diameter of the magnetic particles is less
than 0.1 micrometer, it may take a long time for separation and
purification using magnetism; and if more than 20 micrometers, the
amount of trapped target material such as proteins may be small due
to the surface area becoming smaller.
[0053] Although the internal composition of the magnetic particles
may be homogeneous, most magnetic materials making up the
homogeneous magnetic particles with a particle diameter in the
above-mentioned preferable range are paramagnetic. If repeatedly
separated and purified by magnetism, the magnetic particles may
lose their capability of being redispersed in dispersion media. For
this reason, it is preferable that the magnetic particles have a
heterogeneous internal composition containing fine particles of a
magnetic material exhibiting small residual magnetization. As the
inner structure of the magnetic particles having such a
heterogeneous internal composition, a structure in which the fine
particles of a magnetic material are dispersed in a continuous
phase of a non-magnetic material such as a polymer, a structure
consisting of a secondary aggregate of fine particles of a magnetic
material as a core and a non-magnetic material such as a polymer
layer as a shell, a structure consisting of a non-magnetic material
such as a polymer (non-magnetic nuclear particles) as a core and a
secondary aggregate material of fine particles of a magnetic
material as a shell, and the like can be given. As the polymer
which is included in the magnetic particles, the polymers described
above as the polymer forming the organic polymer particles may be
used. In the case of the structure in which the inner structure
consists of a core of a non-magnetic material such as a polymer
(non-magnetic nuclear particles) and a shell of a secondary
aggregate of fine particles of a magnetic material, it is
preferable that a polymer layer be further formed on the outermost
layer. As the polymer which is used for the outermost layer, the
polymers described above as the polymer forming the organic polymer
particles as the base particles may be used.
[0054] The organic polymer particles of this embodiment may be
produced by a general method such as emulsion polymerization,
soap-free polymerization, and suspension polymerization. When the
organic polymer particles are magnetic particles, such organic
polymer particles may be produced by, for example, mixing the
non-magnetic material nuclear particles with the fine particles of
a magnetic material and causing the fine particles of a magnetic
material to be physically adsorbed on the surface of the
non-magnetic material nuclear particles. In this embodiment,
"physical adsorption" refers to adsorption not involving a chemical
reaction. As the principle of "physical adsorption",
hydrophobic/hydrophobic adsorption, molten bonding or adsorption,
fusion bonding or adsorption, hydrogen bonding, Van der Waals
bonding, and the like can be given, for example.
[0055] More specifically, the organic polymer particles may be
obtained by, for example, suspension polymerization of the above
vinyl monomer or polymer bulk shattering. For example, the organic
polymer particles can be obtained by a two-stage swelling
polymerization method using seed particles described in
JP-B-57-24369, the polymerization method described in J. Polym.
Sci., Polymer Letter Ed., 21, 937 (1963), and the methods described
in JP-A-61-215602, JP-A-61-215603, and JP-A-61-215604.
[0056] The magnetic particles can also be prepared by a method
utilizing hydrophobic/hydrophobic adsorption as mentioned above.
For example, a method selecting non-magnetic nuclear particles and
fine particles of a magnetic material, each having a hydrophobic or
hydrophobized surface, and dry-blending these non-magnetic nuclear
particles and fine particles of a magnetic material, and a method
sufficiently dispersing the non-magnetic nuclear particles and the
fine particles of a magnetic material in a solvent (such as toluene
or hexane) with good dispersibility without damaging both
particles, followed by vaporization of the solvent while mixing can
be given. Alternatively, the magnetic particles may be produced by
a method realizing complexing on non-magnetic nuclear particles and
fine particles of a magnetic material by physically applying a
strong external force. As examples of the method for physically
applying a strong force, a method using a mortar, an automatic
mortar, or a ball mill; a blade-pressuring type powder compressing
method; a method utilizing a mechanochemical effect such as a
mechanofusion method; and a method using an impact in a high-speed
air stream such as a jet mill, a hybridizer, or the like can be
given. In order to efficiently produce a firmly bound complex, a
strong physical adsorption force is desirable. As the method,
stirring using a vessel equipped with a stirrer at a peripheral
velocity of stirring blades of preferably 15 msec or more, more
preferably 30 msec or more, and still more preferably from 40 to
150 msec can be given. If the stirring blade peripheral velocity is
less than 15 msec, sufficient energy for causing fine particles of
a magnetic material to be adsorbed on the surface of non-magnetic
nuclear particles may not be obtained. Although there are no
specific limitations to the upper limit of the peripheral speed of
the stirring blades, the upper limit of the peripheral speed is
determined according to the apparatus to be used, energy
efficiency, and the like.
1-1-2. Saccharide
[0057] As examples of the saccharide used for the carrier polymer
particles according to this embodiment, monosaccharides, such as
furanoses such as fructose, arabinose, xylose, ribose, and
deoxyribose, pyranoses such as glucose, mannose, and galactose, and
septanoses; disaccharides such as trehalose, lactose, kojibiose,
nigerose, maltose, isomaltose, sophorose, laminaribiose,
cellobiose, and gentiobiose; and polysaccharides such as starch,
amylose, amylopectin, dextrin, glycogen, cyclodextrin, cellulose,
agarose, alginic acid, inulin, glucomannan, chitin, chitosan, and
hyaluronic acid can be given. In order to cover the surface of
organic polymer particles by the chemical bond of the organic
polymer particles and the saccharide, a polysaccharide with a high
molecular weight is preferable from the viewpoint of coating
efficiency. A saccharide of which at least a part of the functional
group (such as a hydroxyl group, an amino group, and a carboxyl
group) has been modified, such as carboxymethylcellulose and
carboxymethyldextran, may be used. The modification may be made in
multiple stages, if necessary. More preferably, a carboxymethylated
saccharide such as carboxymethylcellulose or carboxymethyldextran
can be used.
1-2. Process for Producing Carrier Polymer Particles
[0058] The process for producing carrier polymer particles of this
embodiment comprises covering the surface of organic polymer
particles having a particle diameter of 0.1 to 20 micrometers with
a saccharide by chemically bonding the organic polymers and the
saccharide.
[0059] In this embodiment, a general chemical reaction may be used
for chemically bonding the organic polymer particles with a
saccharide without any particular limitations.
[0060] FIG. 1 is a diagram showing the process for producing the
carrier polymer particles according to this embodiment. FIG. 2 is a
diagram showing the process for producing the carrier polymer
particles according to this embodiment, wherein a carrier polymer
particle 10 of the embodiment produced by the process shown in FIG.
1 is shown.
[0061] For example, as shown in FIG. 1, the organic polymer
particle 11 used for producing the carrier polymer particles
according to this embodiment may have a plurality of functional
groups 13 (first functional groups) on the surface. The first
functional groups 13 may be functional groups introduced when the
particle shape of the organic polymer particle 11 is formed or
functional groups obtained by converting the functional groups
after the particle shape of the organic polymer particle 11 has
been formed. The conversion of functional groups may be carried out
two or more times. Although not particularly limited, when the
functional group introduced when forming the particle shape of the
organic polymer particle 11 is an epoxy group, an amino group
produced by reacting the epoxy group with a large excess amount of
ammonia or an appropriate diamine compound may be the first
functional group, or when the functional group introduced when
forming the particle shape of the organic polymer particle 11 is a
hydroxyl group, for example, an amino group produced by converting
the hydroxyl group into a tosyl group and reacting the tosyl group
with a large excess amount of an appropriate diamine compound may
be the first functional group 13. For example, in the organic
polymer particles Am-1 to Am-5 respectively obtained in the
later-described Experimental Examples 1 to 3, and Experimental
Examples 5 to 6, the first functional group 13 may be the amino
group.
[0062] The saccharide 12 used for producing the carrier polymer
particles according to this embodiment may have a plurality of
functional groups (second functional groups) 14 in the molecule.
The functional group may be produced by converting the functional
group of a saccharide.
[0063] As the functional group which can be used as the first
functional group 13 and/or the second functional group 14, a
carboxyl group, a hydroxyl group, an epoxy group, an amino group, a
mercapto group, a vinyl group, an allyl group, an acrylic group, a
methacryl group, a tosyl group, an azido group, and the like can be
given. The first functional group 13 and the second functional
group 14 are reactive with each other. Although not particularly
limited, when the first functional group 13 is an epoxy group, for
example, the second functional group 14 may be an amino group, or
when the first functional group 13 is an amino group, for example,
the second functional group 14 may be a carboxyl group.
[0064] It is possible to chemically bond the organic polymer
particle 11 with a saccharide 12 by reacting the first functional
group 13 and the second functional group 14 (see FIG. 2), whereby
the carrier polymer particle 10 according to this embodiment can be
obtained.
[0065] After preparation according to the above-described process,
the carrier polymer particles of this embodiment can be used as
carrier particles after adjusting the pH and washing the surface by
a purification process such as dialysis, ultrafiltration, and
centrifugation, as required.
1-3. Application
[0066] The carrier polymer particles of this embodiment are used as
chemical bonding carrier polymer particles in the drug discovery
field and also as chemical bonding carrier polymer particles for
diagnostic agent.
[0067] More particularly, the carrier polymer particles of this
embodiment can be used for selecting and purifying proteins and the
like exhibiting specific interactions with a chemical compound to
be analyzed by immobilizing such a chemical compound to be analyzed
by chemical bonding and analyzing and/or measuring the specific
interactions using intermolecular interactions with the proteins
and the like.
[0068] In addition, the carrier polymer particles of this
embodiment can also be used as biologically-related material
carrier polymer particles for sensing proteins such as an antibody,
an antigen, an enzyme, and a hormone, nucleic acids such as DNA and
RNA, and biologically-related glycoside compounds (hereinafter
referred to collectively as "biologically-related material") on the
surface of particles by a chemical bonding method.
[0069] The applications of the carrier polymer particles of this
embodiment are not limited to the above-mentioned applications of
chemical bonding carrier polymer particles in the drug discovery
field and chemical bonding carrier polymer particles for diagnostic
drugs. The carrier polymer particles can be used in a wide variety
of fields such as biologically-related fields, paints, papers,
electrophotography, cosmetics, medical supplies, agricultural
chemicals, foods, and catalysts.
2. Second Embodiment
2-1. Process for Producing Carrier Polymer Particles
[0070] The process for producing carrier polymer particles
according to the second embodiment comprises covering organic
polymer particles with a particle diameter of 0.1 to 20
micrometers, which has a functional group having reactivity with a
carboxyl group, with a saccharide having a carboxyl group by
chemically bonding the organic polymer particles and the saccharide
(a first step), and treating the organic polymer particles, of
which the surface has been covered with the saccharide, with a
basic solution (a second step).
[0071] The first step and second step in the process for producing
the carrier polymer particles according to the embodiment will be
explained in detail.
[0072] 2-1-1. First Step
[0073] The functional group which has reactivity with a carboxyl
group which exists in the organic polymer particles in the first
step of this embodiment is at least one functional group selected
from the group consisting of an amino group, a hydroxyl group, and
an epoxy group, preferably at least one functional group selected
from the group consisting of an amino group and a hydroxyl group,
and more preferably an amino group.
[0074] In the first step of this embodiment, a general chemical
reaction may be used for chemically bonding the organic polymer
particles to a saccharide without any particular limitations. The
particles may be chemically bonded to the saccharide by a bonding
group including at least one of an amide bond and an ester
bond.
[0075] FIG. 1 is a diagram showing the first step of the process
for producing carrier polymer particles according to this
embodiment, and FIG. 2 is a diagram showing a example of the
carrier polymer particles produced in the first step.
[0076] For example, as shown in FIG. 1, the organic polymer
particle 11 used for producing the carrier polymer particles
according to this embodiment has functional groups 13, which are
reactive with a carboxyl group, on the surface. The functional
groups 13 which are reactive with a carboxyl group may be
introduced when the particle shape of the organic polymer particle
11 is formed, or may be obtained by converting a certain group
after the particle shape of the organic polymer particle 11 has
been formed. The conversion of functional groups may be carried out
two or more times. For example, when the functional group
introduced when forming the shape of the organic polymer particle
11 is an epoxy group, an amino group produced by reacting the epoxy
group with a large excess amount of ammonia or an appropriate
diamine compound may be the functional groups 13 which are reactive
with a carboxyl group, or when the functional group introduced when
forming the shape of the organic polymer particle 11 is a hydroxyl
group, an amino group produced by converting the hydroxyl group
into a tosyl group and reacting the tosyl group with a large excess
amount of an appropriate diamine compound may be the functional
groups 13 which are reactive with a carboxyl group. For example, in
the organic polymer particles Am-1 to Am-5 respectively obtained in
the later-described Experimental Examples 1 to 3, and Experimental
Examples 5 to 6, the functional group 13 which is reactive with a
carboxyl group is the amino group.
[0077] The saccharide 12 used for producing the carrier polymer
particles according to this embodiment may have one carboxyl group
14 or a plurality of carboxyl groups 14 in the molecule. The
carboxyl group 14 may be a group produced by converting a specific
functional group of the saccharide 12.
[0078] As an example of the group which can be used as the
functional group 13 reactive with a carboxyl group, an amino group
can be given. As mentioned above, the amino group may be converted
from another group (such as a hydroxyl group or an epoxy group)
which is introduced when the particle shape of the organic polymer
particles are formed. The functional group 13 reactive with a
carboxyl group and the carboxyl group 14 are reactive with each
other. Although not limited, when the functional group 13 having
reactivity with a carboxyl group is at least one selected from the
group consisting of an amino group, a hydroxyl group, and an epoxy
group, for example, this functional group 13 has reactivity with
the carboxyl group 14.
[0079] In FIG. 1, the organic polymer particle 11 and the
saccharide 12 can chemically bond by reacting the functional group
13 having reactivity with a carboxyl group and the carboxyl group
14.
[0080] A general method can be used for this chemical bonding
without specific limitations. For example, when the group having
reactivity with a carboxyl group is an epoxy group, an ester can be
produced by directly reacting them. When the group having
reactivity with a carboxyl group is a hydroxyl group, an
esterification method using various condensing agents can be used
(The 4th edition of Experimental Chemistry Lecture Vol. 22, pp
45-47, 1992). When the group having reactivity with a carboxyl
group is an amino group, an amidation method using various
condensing agents commonly used in organic synthesis (The 4th
edition of Experimental Chemistry Lecture Vol. 22, pp 139-144,
1992) and various methods used for forming a peptide bond in
peptide synthesis (The 4th edition of Experimental Chemistry
Lecture Vol. 22, pp 259-271, 1992) can be used.
[0081] After the organic polymer particle 11 and the saccharide 12
have been chemically bonded and the surface of the organic polymer
particle 11 is covered with the saccharide 12 (see FIG. 2), an
excess amount of the saccharide existing in the reaction system
(not shown in the drawing) may be physically adsorbed in the
saccharide 12 which chemically bonded to the organic polymer
particle 11 by hydrogen bonds and the like between carboxyl group
and carboxyl group, carboxyl group and hydroxyl group, and/or
hydroxyl group and hydroxyl group. In order to sufficiently cover
the surface of the organic polymer particle 11 with a chemically
bonded saccharide 12, it is necessary to use an excess amount of a
saccharide. Therefore, occurrence of physical adsorption of
saccharide 12 to a certain degree is inevitable. If the target
material is separated and purified by using the organic polymer
particle 11 in which a saccharide is physically adsorbed, problems
such as reduced utilization efficiency of the carboxyl group 14, an
increase in nonspecific adsorption due to the surface of the
particle 10 partially made porous, and detachment of the
once-trapped target material together with the physically adsorbed
saccharide during separation and purification operations may
occur.
2-1-2. Second Step
[0082] In the second step of this embodiment, the saccharide
physically adsorbed on the surface of the organic polymer particle
11 in the first step can be extracted by a basic solution by
treating the organic polymer particle 11 of which the surface was
covered by saccharide 12 with the basic solution. In the second
step, by treating the organic polymer particle 11 with a sufficient
amount of basic solution, only chemically bonded saccharide 12
finally remains on the surface of the organic polymer particle 11
(see FIG. 2), whereby the above-mentioned problems caused by the
physically adsorbed saccharide can be overcome. The carrier polymer
particles comprising organic polymer particle 11 and the saccharide
12 covering the surface of the organic polymer particle 11, from
which physically adsorbed saccharide has been removed, can be
obtained by the above steps.
[0083] The basic solution used here is not particularly limited
insofar as the solution can extract the physically adsorbed
saccharide. For example, alkaline aqueous solutions such as a
sodium hydroxide aqueous solution, a potassium hydroxide aqueous
solution, a lithium hydroxide aqueous solution, a sodium carbonate
aqueous solution, a sodium hydrogen carbonate aqueous solution, a
potassium carbonate aqueous solution, a potassium hydrogen
carbonate aqueous solution, a lithium carbonate aqueous solution,
ammonia water, and a hydroxyl tetramethylammonium aqueous solution;
and aqueous solutions of water-soluble organic amines can be
given.
[0084] The concentration of the basic aqueous solution used here is
usually 0.001 M or more. The treating temperature is usually 0 to
50.degree. C., and preferably 0 to 30.degree. C.
[0085] Although inferior to the basic solution in respect of
efficiency, the physically-adsorbed saccharide can also be
extracted by treating with an appropriate electrolytic
solution.
2-1-3. Third Step
[0086] The process for producing the carrier polymer particles may
further comprise a step of chemically bonding the saccharide to a
probe for specifically trapping a target material (a third step).
The particles obtained by the third step have a probe to
specifically trap a target material chemically bonded to a
saccharide (such particles are herein referred to as "probe-bonded
particles). Although not specifically limited, the saccharide and
the probe may bond via a chemical bond such as an --O-- bond, an
--S-- bond, an --SO-- bond, an --SO.sub.2-- bond, a --CO-- bond, a
--CO.sub.2-- bond, an --NR.sup.1-- bond (wherein R.sup.I is an
alkyl group or H), an --N.sup.+R.sup.2R.sup.3-- bond (wherein
R.sup.2 and R.sup.3 are individually an alkyl group or H), an
--NHCO-- bond, or a --PO.sub.2-- bond. The saccharide and the probe
can be chemically bonded by, for example, chemically reacting a
functional group in the saccharide with a functional group in the
probe.
[0087] The functional group in the saccharide and the functional
group in the probe are not specifically limited. As examples,
groups such as a hydroxyl group, an acyl group, a mercapto group,
an amino group, an aminoacyl group, a carbonyl group, a formyl
group, a carboxyl group, an amide group, a sulfonic group, a
phosphate group, an epoxy group, a tosyl group, an azido group, a
vinyl group, and an allyl group can be given.
[0088] In this embodiment, the term "target material" refers to a
target to be trapped by the probe-bonded particles according to
this embodiment. As an example of the target material, a
biologically-related material can be given. In the embodiment, the
term "biologically-related material" refers to all materials
relating to biological bodies. As examples of the
biologically-related material, materials contained in biological
bodies, materials derived from materials contained in biological
bodies, and materials which can be used in biological bodies can be
given.
[0089] More specific examples of the biologically-related materials
include, but are not limited to, proteins (such as an enzyme, an
antibody, and an acceptor), peptides (such as glutathione and RGD
peptides), nucleic acids (such as DNA and RNA), carbohydrates,
lipids, and other cells and materials (such as various
blood-originating materials and various floating cells containing
various blood cells such as platelets, erythrocytes, and
leukocytes).
[0090] When the probe is a protein, for example, the probe can be
chemically bonded to the saccharide by, for example, reacting a
functional group in the protein (for example, an amino group or a
carboxyl group) with a functional group in the saccharide (for
example, a carboxyl group, a hydroxyl group, or an amino group). In
this instance, the probe and the saccharide can be bonded through
an amide bond or an ester bond. When the probe is a nucleic acid,
for example, the probe can be chemically bonded to the saccharide
by, for example, reacting a functional group in the nucleic acid
(for example, a phosphoric acid group) with a functional group in
the saccharide (for example, a hydroxyl group). In this instance,
the probe and the saccharide can be bonded through a phosphodiester
bond.
[0091] Although not particularly limited, the probe which can be
used with the particles for specific trapping includes, for
example, a protein (for example, an antibody, an antigen, an
enzyme, an acceptor, and a hormone), a peptide, a nucleic acid (for
example, DNA and RNA), a glycoside compound, and a synthetic
chemical material (for example, a pharmaceutical candidate
compound).
[0092] When the probe is an antibody (or an antigen), the target
material may be an antigen (or an antibody) which specifically
bonds to the antibody (or the antigen).
[0093] When the probe is a nucleic acid, the target material may be
a nucleic acid which specifically bonds to the nucleic acid. When
the probe is an enzyme, an acceptor, or a hormone, the target
material may be a chemical compound which specifically bonds to the
enzyme, the acceptor, or the hormone.
2-1-4. Materials Used for Producing Carrier Polymer Particles of
this Embodiment
[0094] Next, the materials used for producing the carrier polymer
particles according to this embodiment are explained in detail.
2-1-4A. Organic Polymer Particles
[0095] The average particle diameter of the organic polymer
particles used for producing the carrier polymer particles of this
embodiment is 0.1 to 20 micrometers, more preferably 0.3 to 15
micrometers, and most preferably 1 to 10 micrometers. Also, the
coefficient of variation of the organic polymer particles used in
the embodiment is normally 30% or less, preferably 20% or less, and
more preferably 10% or less.
[0096] In this embodiment, the organic polymer particles may be
used as base particles of the carrier polymer particles produced
according to this embodiment. Organic polymer particles are
suitable as base particles, since it is easy to cover the surface
of organic polymer particles with a saccharide which is bonded by
chemical bonding. Also, magnetic particles may be used as the
organic polymer particles.
[0097] As explained above, when the carrier polymer particles
produced according to this embodiment are dispersed in a solvent,
nonspecific adsorption of biologically-related materials such as
proteins increases if the organic polymer particles are dispersed
in a dispersion medium or the organic polymer particles swell by
the solvent. For this reason, it is desirable that the organic
polymer particles do not dissolve in the dispersion medium. An
aqueous medium may be used as the dispersion medium, for example.
The aqueous medium means water or a mixture of water and a solvent
which dissolves in water (for example, alcohols and alkylene glycol
derivatives).
[0098] As the organic polymer particles, polymers used for the
organic polymer particles of the first embodiment can be used. In
addition, the process described in the first embodiment can be
used. Vinyl polymers are particularly preferable as the polymer
constituting the organic polymer particles.
[0099] As the magnetic particles, those given as the magnetic
particles in first embodiment may be used.
2-1-4B. Saccharide
[0100] As examples of the saccharide having a carboxyl group used
for producing the carrier polymer particles according to this
embodiment, saccharides obtained by chemically modifying at least a
part of the functional groups (for example, a hydroxyl group and an
amino group) in the molecule of a saccharide by introducing a
carboxyl group, for example, carboxymethylcellulose,
carboxymethyldextran, and polysaccharides originally possessing a
carboxyl group (such as alginic acid and hyaluronic acid) can be
given. Such a saccharide to be modified by introducing a carboxyl
group includes furanoses such as fructose, arabinose, xylose,
ribose, and deoxyribose; pyranoses such as glucose, mannose, and
galactose; monosaccharides such as septanoses; disaccharides such
as trehalose, lactose, kojibiose, nigerose, maltose, isomaltose,
sophorose, laminaribiose, cellobiose, and gentiobiose; and
polysaccharides such as starch, amylose, amylopectin, dextrin,
glycogen, cyclodextrin, cellulose, agarose, inulin, glucomannan,
chitin, and chitosan.
[0101] A general chemical reaction may be used for chemically
modifying the saccharide to introduce a carboxylic group without
any particular limitations. The chemical modification may be
carried out in two or more stages as required. In order to cover
the surface of organic polymer particles by the chemical bond of
the organic polymer particles and the saccharides, a polysaccharide
with a high molecular weight is preferable from the viewpoint of
coating efficiency. Carboxymethylcellulose and carboxymethyldextran
are particularly preferable as the saccharide having a carboxyl
group.
2-1-5. Carrier Polymer Particles
[0102] The particle diameter of the carrier polymer particles
produced by this embodiment is preferably 0.1 to 17 micrometers,
and more preferably 1 to 10 micrometers. When the particle diameter
is less than 0.1 micrometer, since separation using centrifugal
separation or the like takes a long time and separation of the
washing solvent such as water and the particles is insufficient,
there are situations in which the removal of non-target molecules
(for example, biologically-related materials such as proteins) is
insufficient and thus, sufficient purification is not possible. In
contrast, since particles with a diameter exceeding 17 micrometers
reduces the surface area of the particles, there may be situations
in which the trapped amount of the biologically-related material
such as a protein, which is the target, is small.
[0103] After preparation according to the above-described process,
the carrier polymer particles of this embodiment can be used as
carrier particles after adjusting the pH and washing the surface by
purification processing such as dialysis, ultrafiltration, and
centrifugation, as required.
[0104] Although it is possible to use the carrier polymer particles
produced by this embodiment as they are, they can also be used as a
dispersion liquid in which the particles are dispersed in a
dispersion medium in order to efficiently carry out a reaction with
a compound. As a dispersion medium, those given as the dispersion
medium in first embodiment may be used.
[0105] In addition, the carrier polymer particles of the embodiment
may be probe-bonded particles in which a probe to specifically trap
a target material chemically bonds to the saccharide. The probe may
be chemically bonded to the saccharide by the above-described third
step. The particle diameter of the probe-bonded particles is
preferably from 0.1 to 20 micrometers, more preferably from 0.3 to
17 micrometers, and still more preferably from 0.5 to 10
micrometers.
2-2. Application
[0106] The carrier polymer particles produced by this embodiment
may be used as chemical bonding carrier polymer particles in the
drug discovery field and also as chemical bonding carrier polymer
particles for diagnostic drugs.
[0107] More particularly, the carrier polymer particles produced by
this embodiment can be used for selecting and purifying a target
material (such as proteins) exhibiting specific interactions with a
chemical compound to be analyzed by immobilizing the chemical
compound to be analyzed by chemical bonding.
[0108] In addition, the carrier polymer particles produced by this
embodiment can also be used as biologically-related material
carrier polymer particles for sensing proteins such as an antibody,
an antigen, an enzyme, and a hormone; nucleic acids such as DNA and
RNA; and biologically-related glycoside compounds (hereinafter
referred to collectively as "biologically-related material") on the
surface of particles by a chemical bonding method.
[0109] The applications of the carrier polymer particles produced
by this embodiment is not limited to the above-mentioned
applications of chemical bonding carrier polymer particles in the
drug discovery field and chemical bonding carrier polymer particles
for diagnostic drugs. The carrier polymer particles can be used in
a wide variety of fields such as biologically-related fields,
paints, papers, electrophotography, cosmetics, medical supplies,
agricultural chemicals, foods, and catalysts.
3. Third Embodiment
3-1. Magnetic Particles for Specific Trapping
[0110] The magnetic particles for specific trapping according to
the third embodiment of the invention comprise magnetic particles
and a saccharide. The saccharide may cover the surface of the
magnetic particles.
[0111] The magnetic particles chemically bond with the saccharide
in the magnetic particles for specific trapping. Although not
specifically limited, the chemical bond of the magnetic particles
and the saccharide is preferably based on a bonding group
containing at least an amide bond or an ester bond.
[0112] In addition, a probe to specifically trap the target
material chemically bonds with the saccharide in the magnetic
particles for specific trapping of this embodiment. Although not
specifically limited, the saccharide and the probe may bond via a
chemical bond such as an --O-- bond, an --S-- bond, an --SO-- bond,
an --SO.sub.2-- bond, a --CO-- bond, a --CO.sub.2-- bond, an --NR--
bond (wherein R is an alkyl group or H), an
--N.sup.+R.sup.2R.sup.3-- bond (wherein R.sup.2 and R.sup.3 are
individually an alkyl group or H), an --NHCO-- bond, or a
--PO.sub.2-- bond. The saccharide and the probe can be chemically
bonded by, for example, chemically reacting a functional group in
the saccharide with a functional group in the probe.
[0113] The functional group in the saccharide and the functional
group in the probe are not specifically limited. As examples,
groups such as a hydroxyl group, an acyl group, a mercapto group,
an amino group, an amino acyl group, a carbonyl group, a formyl
group, a carboxyl group, an amide group, a sulfonic group, a
phosphate group, an epoxy group, a tosyl group, an azido group, a
vinyl group, and an allyl group can be given.
[0114] In this embodiment, the term "target material" refers to a
target to be trapped by the magnetic particles for specific
trapping according to this embodiment. As an example of the target
material, a biologically related material can be given. In the
embodiment, the term "biologically-related material" refers to all
materials relating to biological bodies. As examples of the
biologically-related material, materials contained in biological
bodies, materials derived from materials contained in biological
bodies, and materials which can be used in biological bodies can be
given.
[0115] More specific examples of the biologically-related materials
include, but are not limited to, proteins (e.g., enzymes,
antibodies, and acceptors), peptides (e.g., glutathione, RGD
peptides), nucleic acids (e.g., DNA and RNA), carbohydrates,
lipids, and other cells and substances (e.g., various
blood-originating substances and various floating cells containing
various blood cells such as platelets, erythrocytes, and
leukocytes).
[0116] When the probe is a protein, for example, the probe can be
chemically bonded to a saccharide by, for example, reacting a
functional group in the protein (for example, an amino group and a
carboxyl group) with a functional group in the saccharide (for
example, a carboxyl group, a hydroxyl group, and an amino group).
In this instance, the probe and the saccharide can be bonded
through an amide bond or an ester bond.
[0117] When the probe is a nucleic acid, for example, the probe can
be chemically bonded to a saccharide by, for example, reacting a
functional group in the nucleic acid (for example, a phosphoric
acid group) with a functional group in the saccharide (for example,
a hydroxyl group). In this instance, the probe and the saccharide
can be bonded through a phosphodiester bond.
[0118] Although not particularly limited, the probe which can be
used with the particles for specific trapping includes, for
example, a protein (for example, an antibody, an antigen, an
enzyme, an acceptor, and a hormone), a peptide, a nucleic acid (for
example, DNA and RNA), a glycoside compound, and a synthetic
chemical substance (for example, a pharmaceutical candidate
compound).
[0119] When the probe is an antibody (or an antigen), the target
material may be an antigen (or an antibody) which specifically
bonds to the antibody (or the antigen).
[0120] When the probe is a nucleic acid, the target material may be
a nucleic acid which specifically bonds to the nucleic acid. When
the probe is an enzyme, an acceptor, or a hormone, the target
material may be a chemical compound which specifically bonds to the
enzyme, the acceptor, or the hormone.
[0121] Although the magnetic particles for specific trapping
according to this embodiment can be used as they are, in order to
efficiently perform a reaction with a compound, it is possible to
use them dispersed in a dispersion medium. As a dispersion medium,
those given as the dispersion medium in first embodiment may be
used.
[0122] The particle diameter of the magnetic particles for specific
trapping according to this embodiment is from 0.1 to 20
micrometers, preferably from 0.3 to 17 micrometers, and more
preferably from 0.5 to 10 micrometers. If the particle diameter is
less than 0.1 micrometer, it takes a long time for separation using
magnetism or the like, resulting in insufficient separation of the
particles from a washing solvent such as water. This makes it
difficult to sufficiently remove materials other than the target
material, giving rise to inadequate purification. On the other
hand, if the particle diameter is more than 20 micrometers, the
amount of the target material which can be trapped may be small due
to the surface area becoming smaller.
[0123] Each of the components of the magnetic particles for
specific trapping of this embodiment are described below.
3-1-1. Magnetic Particles
[0124] The average particle diameter of the nuclear particles used
in this embodiment is preferably from 0.1 to 20 micrometers, more
preferably from 0.3 to 17 micrometers, and still more preferably
from 0.5 to 10 micrometers. If the particle diameter of the
magnetic particles is less than 0.1 micrometer, it may take a long
time for separation and purification using magnetism; and if more
than 20 micrometers, the amount of trapped target material may be
small due to the surface area becoming smaller.
[0125] As mentioned above, when the magnetic particles for specific
trapping of this embodiment are dispersed in a solvent, the
nonspecific adsorption of the target materials increases if the
magnetic particles are dissolved in the dispersion medium or the
magnetic particles are swollen by the solvent. For this reason, it
is desirable that the magnetic particles are not dissolved in a
solvent.
[0126] Although the internal composition of the magnetic particles
used in this embodiment may be homogeneous, most magnetic materials
making up the homogeneous magnetic particles with a particle
diameter in the above-mentioned preferable range are paramagnetic.
If repeatedly separated and refined by magnetism, the magnetic
particles may lose their capability of being dispersed in
dispersion media. For this reason, it is preferable that the
magnetic particles of this embodiment have a heterogeneous internal
composition containing fine particles of a magnetic material
exhibiting small residual magnetization. As the inner structure of
the magnetic particles having such a heterogeneous internal
composition, (i) a structure in which the magnetic particles are
dispersed in a continuous phase of a non-magnetic material such as
a polymer, (ii) a structure consisting of a secondary aggregate of
fine particles of a magnetic material as a core and a non-magnetic
material such as a polymer layer as a shell, and (iii) a structure
consisting of a non-magnetic material such as a polymer
(non-magnetic nuclear particles) as a core and a magnetic material
layer (secondary aggregate materials of fine particles of a
magnetic material) of supermagnetic nanoparticles provided on the
surface of the nuclear particles, and the like can be given. As the
polymer which can be used as the core, polymers described later as
the polymer forming the magnetic particles may be used. The fine
particles of a magnetic material in the above structures (i) to
(iii) are preferably fine particles of at least one of
Fe.sub.2O.sub.3 and Fe.sub.3O.sub.4.
[0127] In the case of the structure of (iii) above, in which the
inner structure consists of a core of a non-magnetic material such
as a polymer (non-magnetic nuclear particles) and a shell of a
magnetic material layer (a secondary aggregate of fine particles of
a magnetic material), it is preferable that a polymer layer be
further formed on the magnetic material layer. In this instance,
the polymer layer may be formed by polymerization on the surface of
the mother particles which contain nuclear particles (core) and a
magnetic material layer (shell) formed on the surface of the
nuclear particles. The magnetic material layer (shell) may contain
fine particles of a magnetic material which contain at least one of
Fe.sub.2O.sub.3 and Fe.sub.3O.sub.4. As the polymer which can be
used for the polymer layer, polymers described later as the polymer
forming the magnetic particles may be used.
[0128] In the case of the above structure (iii), the magnetic
material layer may be produced by, for example, mixing the
non-magnetic material nuclear particles with the fine particles of
a magnetic material and causing the fine particles of a magnetic
material to be physically adsorbed on the surface of the
non-magnetic material nuclear particles. In this embodiment,
"physical adsorption" refers to adsorption not involving a chemical
reaction. As the principle of "physical adsorption",
hydrophobic/hydrophobic adsorption, molten bonding or adsorption,
fusion bonding or adsorption, hydrogen bonding, Van-der-Waals
bonding, and the like can be given, for example.
[0129] The magnetic particles of the structure (iii) above can be
obtained by, for example, suspension polymerization of the above
vinyl monomer or polymer bulk shattering. For example, the magnetic
particles can be obtained by the two-stage swelling polymerization
method using seed particles described in JP-UM-B-57-24369, the
polymerization method described in J. Polym. Sci., Polymer Letter
Ed., 21, 937 (1963), and the methods described in JP-A-61-215602,
JP-A-61-215603, and JP-A-61-215604.
[0130] The magnetic particles of the structure (iii) above can also
be prepared by a method utilizing hydrophobic/hydrophobic
adsorption. For example, a method selecting non-magnetic nuclear
particles and fine particles of a magnetic material, each having a
hydrophobic or hydrophobized surface, and dry-blending these
non-magnetic nuclear particles and fine particles of a magnetic
material, and a method sufficiently dispersing the non-magnetic
nuclear particles and fine particles of a magnetic material in a
solvent (such as toluene or hexane) with good dispersibility
without damaging both particles, followed by vaporization of the
solvent while mixing can be given.
[0131] Alternatively, in the case of the above structure (iii), the
magnetic particles may be produced by physically applying a strong
external force to cause the fine particles of a magnetic material
to be adsorbed on the surface of the non-magnetic material nuclear
particles. As examples of the method for physically applying a
strong force, a method using a mortar, an automatic mortar, or a
ball mill; a blade-pressuring type powder compressing method; a
method utilizing a mechanochemical effect such as a mechanofusion
method; and a method using an impact in a high-speed air stream
such as a jet mill, a hybridizer, or the like can be given. In
order to efficiently produce a firmly bound complex, a strong
physical adsorption force is desirable. As the method, stirring
using a vessel equipped with a stirrer at a peripheral velocity of
stirring blades of preferably 15 m/sec or more, more preferably 30
m/sec or more, and still more preferably from 40 to 150 m/sec can
be given. If the stirring blade peripheral velocity is less than 15
m/sec, sufficient energy for causing fine particles of a magnetic
material to be adsorbed on the surface of non-magnetic nuclear
particles may not be obtained. Although there are no specific
limitations to the upper limit of the peripheral speed of the
stirring blades, the upper limit of the peripheral speed is
determined according to the apparatus to be used, energy
efficiency, and the like.
[0132] As the polymer used for forming the magnetic particles of
this embodiment, those mentioned in connection with the organic
polymer particles of the first embodiment can be given.
3-1-2. Saccharide
[0133] As the saccharide used for forming the magnetic particles
for specific trapping of this embodiment, those mentioned in the
first embodiment can be given.
3-2. Process for Producing Magnetic Particles for Specific
Trapping
[0134] The process for producing the magnetic particles for
specific trapping according to this embodiment comprises chemically
bonding magnetic particles with a diameter of 0.1 to 20 micrometers
with a saccharide and chemically bonding a probe for specifically
trapping the target material with a saccharide. The surface of the
magnetic particles can be covered with a saccharide by chemically
bonding the magnetic particles with the saccharide.
[0135] In this embodiment, a general chemical reaction may be used
for chemically bonding the magnetic particles with the saccharide
without any particular limitations. For example, the magnetic
particles used for producing the magnetic particles for specific
trapping according to this embodiment may have two or more
functional groups (first functional groups) on the surface. The
first functional group may be a functional group introduced when
the particle shape of magnetic particles is formed or a functional
group obtained by converting the functional group after the
particle shape of magnetic particles has been formed. The
conversion of functional groups may be carried out two or more
times. Although not particularly limited, when the functional group
introduced when forming the particle shape of the magnetic
particles is an epoxy group, an amino group produced by reacting
the epoxy group with a large excess amount of ammonia or an
appropriate diamine compound may be the first functional group, or
when the functional group introduced when forming the particle
shape of the magnetic particles is a hydroxyl group, for example,
an amino group produced by converting the hydroxyl group into a
tosyl group and reacting the tosyl group with a large excess amount
of an appropriate diamine compound may be the first functional
group. For example, in the magnetic particles Am-6 and Am-7
respectively obtained in the later-described Experimental Examples
8 and 9, the first functional group can be the amino group.
[0136] The saccharide used for producing the magnetic particles for
specific trapping according to this embodiment may have two or more
functional groups (second functional groups) in the molecule.
[0137] As the functional group which can be used as the first
functional group and/or the second functional group, a carboxyl
group, a hydroxyl group, an epoxy group, an amino group, a mercapto
group, a vinyl group, an allyl group, an acrylic group, a methacryl
group, a tosyl group, an azido group, and the like can be given.
The first functional group and the second functional group are
reactive with each other. Although not particularly limited, when
the first functional group is an epoxy group, for example, the
second functional group may be an amino group, or when the first
functional group is an amino group, for example, the second
functional group may be a carboxyl group.
[0138] It is possible to chemically bond the magnetic particles
with the saccharide by reacting the first functional group and the
second functional group, whereby the magnetic particles for
specific trapping according to this embodiment can be obtained.
[0139] In this embodiment, a general chemical reaction may be used
for chemically bonding the magnetic particles to the saccharide
without any particular limitations. The probe and the saccharide
can be chemically bonded by, for example, chemically reacting a
functional group in the probe with a functional group in the
saccharide. The functional group contained in the probe and the
functional group contained in the saccharide are the groups
mentioned above.
[0140] After preparation according to the above-described process,
the magnetic particles for specific trapping of this embodiment can
be used as carrier particles after adjusting the pH and washing the
surface by purification processing such as dialysis,
ultrafiltration, and centrifugation, as required.
4. Examples
[0141] The invention will now be described in more detail by way of
examples, which should not be construed as limiting the invention.
In the Examples, "%" and "part" are indicated on the weight
basis.
4-1. Example 1
4-1-1. Evaluation Method
4-1-1A. Evaluation 1 of Nonspecific Adsorption (Protein
Adsorption)
4-1-1A-1. Pre-Washing Step
[0142] Carrier polymer particles prepared in the later-described
Experimental Examples and Comparative Examples were diluted with
and dispersed in purified water to obtain dispersion liquids, each
having a particle concentration of 1 wt %. 500 microliters of the
dispersion liquid was put into a microcentrifuge tube ("Safe-Lock
Tube" manufactured by Eppendorf AG) and centrifuged (15,000 rpm,
15.degree. C., 10 minutes) using a centrifugal separator ("MX-150"
manufactured by Tomy Seiki Co.) to remove the supernatant liquid.
500 microliters of a PBS(-) buffer solution was added to the tube
which contained the precipitate, and the mixture was vibrated by a
touch mixer to disperse the particles.
4-1-1A-2. Protein Adsorption Reaction Step
[0143] Then, 500 microliters of a PBS(-) solution of 1 wt % BSA
(bovine serum albumin) was added to the tube and the mixture was
vibrated by a touch mixer to disperse the particles in the
solution, followed by mixing by rotation and inversion for two
hours at room temperature.
4-1-1A-3. Washing Step
[0144] After centrifugal separation, the supernatant liquid was
removed. 1 ml of 10 mM HEPES was added to the tube and the
particles were dispersed by vibration using a touch mixer. After
repeating the same procedure twice, the content was transferred to
another microcentrifuge tube to perform centrifugal separation, and
the supernatant liquid was removed.
4-1-1A-4. Detaching Step
[0145] After the addition of 50 microliters of a 0.5% aqueous
solution of SDS (sodium dodecylsulfate), the mixture was gently
vibrated by a touch mixer to disperse the particles. After allowing
the mixture to stand for 10 minutes, centrifugal separation was
performed and 20 microliters of the supernatant liquid was
collected.
4-1-1A-5. Sampling Step
[0146] 2-mercaptoethanol was dissolved in a premix sample buffer
solution manufactured by Bio-Rad Laboratories, Inc. to a
concentration of 2 wt % (this solution is hereinafter referred to
as "sample buffer"). 20 microliters of the solution was collected
in the microcentrifuge tube. 20 microliters of the supernatant
liquid collected in the above step was mixed and heated at
100.degree. C. for five minutes in a tube heater.
[0147] As controls, a 1 wt % BSA solution in PBS(-) was diluted
with a 2% SDS solution to 5,000 fold, 10,000 fold, and 20,000 fold.
20 microliters of each of the diluted solutions was mixed with 20
microliters of the sample buffer and heated in a tube heated at
100.degree. C. for five minutes. The resulting solutions are called
"reference diluted BSA".
4-1-1A-6. Electrophoresis (SDS-PAGE)
[0148] The reference diluted BSA was applied to a vertical
electrophoresis system ("Mini-PROTEAN3" manufactured by Bio-Rad
Laboratories, Inc.) in an amount of 20 microliters per one lane of
the gel to perform electrophoresis using a precast polyacrylamide
gel ("Ready Gel J" (15%) manufactured by Bio-Rad Laboratories,
Inc.) and a premix electrophoresis buffer solution manufactured by
Bio-Rad Laboratories, Inc. The gel was stained by a standard
staining method using "Silver Stain Plus Kit" manufactured by
Bio-Rad Laboratories, Inc. An image was produced by scanning the
stained gel using a densitometer "GS-700" manufactured by Bio-Rad
Laboratories, Inc. and the product of the concentration and the
area of the BSA band in the gel was determined using an analysis
software "Multi-Analyst".
[0149] Since the weight of BSA which flows per one lane of the gel
is known in the reference dilution BSA, a calibration curve was
drawn from the product of the band concentration and the area, and
the amount of BSA detached from the particles was converted on a
weight basis based on the calibration curve. The resulting weight
corresponded to the amount of BSA which had been adsorbed per 1 mg
of the particles.
4-1-1B. Particle Diameter
[0150] The diameter of the particles with a diameter of 1
micrometer or more was measured using a laser diffraction particle
size distribution analyzer ("SALD-200V" manufactured by Shimadzu
Corp.) and the diameter of the particles with a diameter of less
than 1 micrometer was measured using a particle size distribution
analyzer based on a laser dispersion diffraction method ("LS 13
320" manufactured by Beckmann Coulter).
4-1-1C. Infrared Absorption Spectrum
[0151] The infrared absorption spectrum was measured by a KBr
method using a Fourier-transform infrared spectrophotometer
("JIR-5500" manufactured by JEOL Ltd.).
4-1-2. Synthesis Examples
4-1-2A. Synthesis Example 1
Synthesis of Organic Polymer Particles A-1
[0152] The organic polymer particles A-1 were prepared by a
two-step swelling polymerization method using seed particles.
[0153] Using polystyrene particles with a particle diameter of 0.98
micrometers obtained by soap-free polymerization as seed particles,
a water dispersion (solid content: 5.0 g) was prepared by
dispersing these polystyrene particles in 500 g of water in a
nitrogen atmosphere. According to the two step swelling
polymerization method (based on the method described in
JP-B-57-24369), an organic solvent (0.1 g of "Shellsol TK") was
added to the seed particles as a first step and monomers (70 g of
MMA (methyl methacrylate), 10 g of TMP (trimethylolpropane
trimethacrylate), and 20 g of GMA (glycidyl methacrylate)) were
added as a second step to cause them to be adsorbed. Then, 2 g of
AIBN (azobisisobutyronitrile) was added and the mixture was slowly
stirred at 75.degree. C. for 24 hours. The reaction solution was
cooled and filtered through a 500 mesh wire gauze to confirm that
99% of the product passed through the wire gauze. The
polymerization stability was good. The polymerization yield was
99%. The particle diameter of the resulting organic polymer
particles A-1 was 2.71 micrometers, the coefficient of variation of
the particle diameter was 2%, and the particles were monodisperse
particles.
4-1-2B. Synthesis Example 2
Synthesis of Organic Polymer Particles A-2
[0154] Organic polymer particles A-2 with a particle diameter of
2.64 micrometers and a coefficient of variation of 2% were obtained
in the same manner as in Synthetic Example 1, except for using 50 g
of MMA, 10 g of TMP, and 40 g of GMA as monomers.
4-1-2C. Synthesis Example 3
Synthesis of Organic Polymer Particles A-3
[0155] Organic polymer particles A-3 with a particle diameter of
2.61 micrometers and a coefficient of variation of 2.1% were
obtained in the same manner as in Synthetic Example 1, except for
using 30 g of MMA, 10 g of TMP, and 60 g of GMA as monomers.
4-1-2D. Synthesis Example 4
Synthesis of Organic Polymer Particles A-4
[0156] Organic polymer particles A-4 with a particle diameter of
7.05 micrometers and a coefficient of variation of 2.3% were
obtained in the same manner as in Synthetic Example 3, except for
using polystyrene particles with a particle diameter of 2.6
micrometers as seed particles.
4-1-2E. Synthesis Example 5
Synthesis of Organic Polymer Particles A-5
[0157] Organic polymer particles A-5 with a particle diameter of
2.58 micrometers and a coefficient of variation of 2.3% were
obtained in the same manner as in Synthetic Example 1, except for
using 10 g of TMP and 90 g of GMA as monomers.
4-1-2F. Synthesis Example 6
Synthesis of Saccharide CMC-1
[0158] Diluted hydrochloric acid was added to an aqueous solution
of carboxymethylcellulose sodium salt ("APP-84" manufactured by
Nippon Paper Chemicals Co., Ltd., a compound having an average
molecular weight of 17,000 and an average of 0.7 carboxyl groups
per one glucose unit) until the solution has a pH of 2 or less. The
resulting solution was dialyzed and concentrated to obtain a 2.5%
aqueous solution of carboxymethylcellulose CMC-1.
4-1-2G Synthesis Example 7
Synthesis of Saccharide CMD-1
[0159] 0.72 g of sodium hydroxide and 1.04 g of bromoacetic acid
were added to 2.5 g of a 10 wt % aqueous solution of Dextran T500
(average molecular weight: 500,000) manufactured by Pharmacia AB,
and the mixture was stirred for several minutes until homogenized.
The solution was maintained at 40.degree. C. for 60 hours and then
cooled with ice. After the addition of diluted hydrochloric acid to
make the pH 2 or less, the solution was dialyzed and freeze-dried
to obtain carboxymethyldextran CMD-1. Carboxylic acid contained in
CMD-1 was measured by titration to find that CMD-1 contained an
average of 0.4 carboxylic acid groups per one glucose unit.
4-1-3. Experimental Example 1
[0160] The polymer particles isolated from the dispersion liquid of
organic polymer particles A-1 by centrifugation were washed by
dispersing in acetone, followed by centrifugation. This washing
procedure was repeated three times. The resulting particles were
dried. 0.50 g of the particles was put into a 100 ml flask and 25 g
of ethylenediamine was added. The particles were irradiated with
indirect ultrasonic radiation for 10 minutes and dispersed. The
dispersion liquid was stirred at 50.degree. C. in a nitrogen
atmosphere for six hours, followed by isolation of the particles by
centrifugal separation. The particles were washed twice with
methanol and three times with a 3:1 (by volume) mixture of water
and methanol, and dried to obtain 0.54 g of organic polymer
particles Am-1 as a white powder.
[0161] The weight of the organic polymer particles Am-1 was larger
than the weight of the organic polymer particles A-1. Comparison of
the infrared absorption spectrum of the organic polymer particles
Am-1 (after ethylenediamine treatment) with the infrared absorption
spectrum of the organic polymer particles A-1 (before
ethylenediamine treatment) indicates that in the infrared
absorption spectrum of the organic polymer particles Am-1, a peak
originating from an epoxy group, which was observed around 900
cm.sup.-1 of the infrared absorption spectrum of the organic
polymer particles A-1, disappeared and, instead, peaks typical to a
primary amine appeared around 3,300 cm.sup.-1 and 3,500 cm.sup.-1.
The organic polymer particles Am-1 were thus confirmed to have an
amino group introduced into the organic polymer particles A-1. That
is, in the organic polymer particles Am-1, the first functional
group 13 is an amino group.
[0162] 30.6 mg of the organic polymer particles Am-1 was added to
1.2 g of a 2.5% aqueous solution of CMC-1 which was obtained in
Synthetic Example 6. The mixture was irradiated with indirect
supersonic waves for 30 minutes to disperse the polymer particles
in the solution. Next, the dispersion liquid was cooled with ice,
and 0.30 g of a 10 wt % aqueous solution of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was
added. The mixture was stirred under ice cooling for 12 hours. The
particles were isolated by centrifugal separation, dispersed in
purified water, isolated by centrifugal separation, and washed.
This procedure was repeated 10 times, followed by drying to obtain
33.6 mg of carrier polymer particles P-1.
[0163] In addition to the peaks originating from the organic
polymer particles Am-1 before the reaction, peaks originating from
carboxymethylcellulose were observed around 3,400 cm.sup.-1 and
1,600 cm.sup.-1 in the infrared absorption spectrum of the carrier
polymer particles P-1. The carrier polymer particles P-1 were thus
confirmed to have a saccharide (carboxymethylcellulose) bonded to
the organic polymer particles Am-1.
[0164] The nonspecific protein adsorption of the carrier polymer
particles P-1 was measured according the above-described method to
confirm that the value was very low (0.08 ng/mg).
4-1-4. Experimental Example 2
[0165] 0.57 g of organic polymer particles Am-2 were obtained in
the same manner as in Experimental Example 1, except for using a
dispersion liquid of organic polymer particles A-2. Next, 34.0 mg
of carboxymethylcellulose-bonded particles (carrier polymer
particles) P-2 was obtained in the same manner as in Experimental
Example 1, except for using the organic polymer particles Am-2
(29.6 mg) and a 2.5% aqueous solution of CMC-1 (1.2 g).
[0166] The nonspecific protein adsorption of the carrier polymer
particles P-2 was measured according the above-described evaluation
method to confirm that the value was very low (0.05 ng/mg).
4-1-5. Experimental Example 3
[0167] 0.61 g of organic polymer particles Am-3 was obtained in the
same manner as in Experimental Example 1, except for using a
dispersion liquid of the organic polymer particles A-3. 36.2 mg of
carboxymethylcellulose-bonded particles (carrier polymer particles)
P-3 was obtained in the same manner as in Experimental Example 1,
except for using the organic polymer particles Am-3 (29.9 mg) and a
2.5% aqueous solution of CMC-1 (1.2 g).
[0168] The nonspecific protein adsorption of the carrier polymer
particles P-3 was measured according the above-described method to
confirm that the value was very low (0.02 ng/mg).
4-1-6. Experimental Example 4
[0169] 150 mg of CMD-1 which was obtained in Synthetic Example 7
was dissolved in 6 g of purified water. 150.5 mg of the organic
polymer particles Am-1 which were obtained in Experimental Example
1 was added to the solution, and the mixture was irradiated with
indirect supersonic waves for 30 minutes to disperse the particles
in the solution. Next, the dispersion liquid was cooled with ice,
and 1.40 g of a 5 wt % aqueous solution of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was
added. The mixture was stirred under ice cooling for 12 hours. The
particles were isolated by centrifugal separation, dispersed in
purified water, isolated by centrifugal separation, and washed.
This procedure was repeated 10 times to obtain 156.2 mg of carrier
polymer particles P-4.
[0170] In addition to the peaks originating from the organic
polymer particles Am-1 before the reaction, peaks originating from
carboxymethyldextran were observed around 3,400 cm.sup.-1 and 1,600
cm.sup.-1 in the infrared absorption spectrum of the carrier
polymer particles P-4. The carrier polymer particles P-4 were thus
confirmed to have a saccharide (carboxymethyldextran) bonded to the
organic polymer particles Am-1.
[0171] The nonspecific protein adsorption of the carrier polymer
particles P-4 was measured according the above-described evaluation
method to confirm that the value was very low (0.07 ng/mg).
4-1-7. Experimental Example 5
[0172] 0.62 g of organic polymer particles Am-4 was obtained in the
same manner as in Experimental Example 1, except for using a
dispersion liquid of the organic polymer particles A-4. 36.1 mg of
carboxymethylcellulose-bonded particles (carrier polymer particles)
P-5 was obtained in the same manner as in Experimental Example 1,
except for using the organic polymer particles Am-4 (29.9 mg) and a
2.5% aqueous solution of CMC-1 (1.2 g).
[0173] The nonspecific protein adsorption of the carrier polymer
particles P-5 was measured according the above-described evaluation
method to confirm that the value was very low (0.05 ng/mg).
4-1-8. Experimental Example 6
[0174] The polymer particles isolated from the dispersion liquid of
organic polymer particles A-5 by centrifugation were washed by
dispersing in acetone, followed by centrifugation. This washing
procedure was repeated three times. The resulting particles were
dried.
[0175] 0.50 g of the particles was put into a 100 ml flask and 46.5
g of dimethylsulfoxide was added. The mixture was irradiated with
indirect ultrasonic radiation for 10 minutes and, after the
addition of 3.5 g of a 10 wt % ethylenediamine solution (solvent:
dimethylsulfoxide), the mixture was stirred at 50.degree. C. in a
nitrogen atmosphere for four hours, followed by isolation of the
particles by centrifugal separation. The particles were washed
twice with methanol and three times with a 3:1 (by volume) mixture
of water and methanol, and dried to obtain 0.55 g of organic
polymer particles Am-5 as a white powder.
[0176] The weight of the organic polymer particles Am-5 was larger
than the weight of the organic polymer particles A-5. Comparison of
the infrared absorption spectrum of the organic polymer particles
Am-5 (after ethylenediamine treatment) with the infrared absorption
spectrum of the organic polymer particles A-5 (before
ethylenediamine treatment) indicates that in the infrared
absorption spectrum of the organic polymer particles Am-5, a peak
originating from an epoxy group, which was observed around 900
cm.sup.-1 of the infrared absorption spectrum of the organic
polymer particles A-5, disappeared and, instead, peaks typical to a
primary amine appeared around 3,300 cm.sup.-1 and 3,500 cm.sup.-1.
Based on the results, the organic polymer particles Am-5 were
confirmed to have an amino group introduced into the organic
polymer particles A-5. Based on the change in the peak intensity
originating from the epoxy group before and after the treatment
with ethylenediamine, the reaction rate of the epoxy group which
exists in the organic polymer particles A-5 was presumed to be
about 30%.
[0177] 100 mg of the organic polymer particles Am-5 was added to a
mixture of 15 g of 1 wt % sulfuric acid and 1.5 g of acetone. The
mixture was irradiated with indirect ultrasonic radiation for 10
minutes and dispersed. The dispersion liquid was stirred at
50.degree. C. for nine hours, followed by isolation of the
particles by centrifugal separation.
[0178] The particles were washed three times with water, once with
a 0.01 N sodium hydroxide aqueous solution, and five times with
water, and dried to obtain 102 mg of particles.
[0179] Comparison of the infrared absorption spectrum of the
organic polymer particles Am-5 before reaction with the infrared
absorption spectrum of the particles obtained here (after the
treatment with sulfuric acid) indicates that in the infrared
absorption spectrum of the organic polymer particles Am-5,
intensity of a peak originating from an epoxy group, which was
observed around 900 cm.sup.-1 of the infrared absorption spectrum
of the organic polymer particles Am-5, disappeared and, instead, a
peak originating from a hydroxyl group around 3,500 cm.sup.-1 was
intensified. Based on the results, it was confirmed that the epoxy
group in the organic polymer particles Am-5 has been
hydrolyzed.
[0180] 33.1 mg of carboxymethylcellulose-bonded particles (carrier
polymer particles) P-6 was obtained in the same manner as in
Experimental Example 1, except for using 30.3 mg of the particles
obtained above.
[0181] The nonspecific protein adsorption of the carrier polymer
particles P-6 was measured according the above-described method to
confirm that the value was less than the detectable limit (0.01
ng/mg).
4-1-9. Experimental Example 7
[0182] Sodium salt of carboxymethylcellulose ("APP-84" manufactured
by Nippon Paper Industries Chemical, Inc.) was purified by
dialyzing an aqueous solution, followed by freeze-drying. An
experiment was carried out in the same manner as in Experimental
Example 1, except for using the purified APP-84 (33 mg) and the
organic polymer particles Am-7 (29.8 mg). Next, the particles were
isolated by centrifugal separation, dispersed in purified water,
and washed. This procedure was carried out five times, a procedure
of dispersing the particles in a 0.01 N hydrochloric acid solution
was carried out three times, and a procedure of dispersing in
purified water, followed by centrifugal separation was carried out
five times. The resulting particles were dried to obtain 33.7 mg of
carboxymethylcellulose-bonded particles (carrier polymer particles)
P-7.
[0183] The nonspecific protein adsorption of the carrier polymer
particles P-7 was measured according the above-described evaluation
method to confirm that the value was very low (0.05 ng/mg).
4-1-10. Comparative Example 1
[0184] The nonspecific protein adsorption of the organic polymer
particles A-1 was measured according the above-described method to
confirm that the value was high (1.3 ng/mg).
4-1-11. Comparative Example 2
[0185] Commercially available standard polystyrene particles
("STADEX SC200S" manufactured by JSR Corporation) was sufficiently
washed with purified water and their nonspecific protein adsorption
was measured according the above-described method to confirm that
the value was very high (20 ng/mg).
4-1-12. Comparative Example 3
[0186] Particles of which the surface was covered with polyethylene
glycol were obtained in the same manner as in Experimental Example
1, except for using a 2.5% aqueous solution of polyethylene glycol
with both terminals modified with carboxylic acid (the number of
average repetition of ethylene oxide unit: 10) instead of a 2.5%
aqueous solution of CMC-1. The nonspecific protein adsorption of
P-8 was measured according the above-described method to confirm
that the value was 0.3 ng/mg.
4-2. Example 2
4-2-1. Method of Evaluation of Properties
4-2-1A. Particle Diameter
[0187] The diameter of the particles with a diameter of 1
micrometer or more was measured using a laser diffraction particle
size distribution analyzer ("SALD-200V" manufactured by Shimadzu
Corp.) and the diameter of the particles with a diameter of less
than 1 micrometer was measured using a particle size distribution
analyzer based on a laser dispersion diffraction method ("LS 13
320" manufactured by Beckmann Coulter).
4-2-1B. Infrared Absorption Spectrum
[0188] The infrared absorption spectrum was measured by a KBr
method using a Fourier-transform infrared spectrophotometer
("JIR-5500" manufactured by JEOL Ltd.).
]
4-2-2. Synthesis Examples
4-2-2A. Synthesis Example 8
Synthesis of Magnetic Particles A-6
[0189] Referring to the polymerization method described in
JP-A-7-238105, styrene/divinylbenzene (96/4) copolymer particles
(average particle diameter: 1.5 micrometers) were prepared. After
polymerization, the particles were separated by centrifugation,
washed with water, dried, and ground. The ground particles were
used as core particles (a-1) (preparation of core particles).
[0190] Next, ferrite-type fine particles of a magnetic material
(average primary particle diameter: 0.02 micrometers) with a
hydrophobized surface were prepared by adding acetone to an oily
magnetic fluid ("EXP series" manufactured by Ferrotec Corp.) to
obtain a precipitate of the particles and drying the
precipitate.
[0191] Then, 15 g of the above core particles (a-1) and 15 g of the
hydrophobized fine particles of a magnetic material were thoroughly
mixed in a mixer. The mixture was processed by a hybridization
system ("NHS-0 type" manufactured by Nara Machinery Co., Ltd.) at a
peripheral blade (stirring blades) speed of 100 m/sec (16,200 rpm)
for five minutes to obtain particles (1) with a magnetic material
layer of fine particles of a magnetic material (M-1) with a number
average particle diameter of 2.0 micrometers on the surface
(preparation of magnetic material layer).
[0192] A 500 ml separable flask was charged with 375 g of an
aqueous solution containing 0.25 wt % of sodium
dodecylbenzenesulfonate and 0.25 wt % of a nonionic emulsifying
agent ("Emulgen 150" manufactured by Kao Corp.), followed by the
addition of 15 g of the above particles (1) having a magnetic
material layer on the surface. After dispersion using a
homogenizer, the resulting dispersion liquid was heated to
60.degree. C. Next, a pre-emulsion, prepared by dispersing 27 g of
MMA (methyl methacrylate), 3 g of TMP (trimethylolpropane
trimethacrylate), and 0.6 g of di(3,5,5-trimethylhexanoyl) peroxide
("Peroyl 355" manufactured by NOF Corp.) in 150 g of an aqueous
solution containing 0.25 wt % of sodium dodecylbenzenesulfonate and
0.25 wt % of a nonionic emulsifying agent ("Emulgen 150"
manufactured by Kao Corp.), was dripped into the above 500 ml
separable flask controlled at 60.degree. C. over one and a half
hours (a first stage polymerization for polymer layer
formation).
[0193] After completing the dripping, the mixture was maintained at
60.degree. C. while stirring for one hour. Next, a pre-emulsion,
prepared by dispersing 7.5 g of MMA, 6 g of GMA (glycidyl
methacrylate), 1.5 g of TMP, and 0.3 g of
di(3,5,5-trimethylhexanoyl) peroxide ("Peroyl 355" manufactured by
NOF Corp.) in 75 g of an aqueous solution containing 0.25 wt % of
sodium dodecylbenzenesulfonate and 0.25 wt % of a nonionic
emulsifying agent ("Emulgen 150" manufactured by Kao Corp.), was
dripped to the above 500 ml separable flask controlled at
60.degree. C. over one and a half hours (a second stage
polymerization for polymer layer formation). After heating to
75.degree. C., the polymerization was continued for a further two
hours before completing the reaction. The resulting water
dispersion of polymer-covered magnetic particles was purified by
magnetism and gravity precipitation to obtain a water dispersion of
magnetic particles A-6 with a solid component concentration of 1%.
The number average particle diameter of the magnetic particles A-6
was 2.9 micrometers.
4-2-2B. Synthesis Example 9
Synthesis of Magnetic Particles A-7
[0194] A 500 ml separable flask was charged with 225 g of a 0.5 wt
% sodium dodecylbenzenesulfonate aqueous solution. 9 g of the
particles (1) having a magnetic material layer were added and
dispersed using a homogenizer, and the resulting dispersion liquid
was heated to 60.degree. C. A pre-emulsion, prepared by dispersing
16.2 g of MMA, 1.8 g of TMP, and 0.36 g of
di(3,5,5-trimethylhexanoyl)peroxide ("Peroyl 355" manufactured by
NOF Corp.) in 90 g of an aqueous solution containing 0.5 wt % of
sodium dodecylbenzenesulfonate was dripped into the above 500 ml
separable flask controlled at 60.degree. C. over one and a half
hours (a first stage polymerization for polymer layer
formation).
[0195] After completion of dripping, the mixture was maintained at
60.degree. C. for one hour while stirring. A pre-emulsion, prepared
by dispersing 8.1 g of GMA, 0.9 g of TMP, and 0.18 g of
di(3,5,5-trimethylhexanoyl)peroxide ("Peroyl 355" manufactured by
NOF Corp.) in 45 g of an aqueous solution containing 0.5 wt % of
sodium dodecylbenzenesulfonate was dripped into the above 500 ml
separable flask controlled at 60.degree. C. over one and a half
hours (a second stage polymerization for polymer layer formation).
After heating to 75.degree. C., the polymerization was continued
for two hours before completing the reaction. The resulting water
dispersion of polymer-covered magnetic particles was purified by
magnetism and gravity precipitation to obtain a water dispersion of
magnetic particles A-7 with a solid component concentration of 1%.
The number average particle diameter of the magnetic particles A-7
was 2.6 micrometers.
4-2-2C. Synthesis Example 10
Synthesis of Saccharide CMC-1
[0196] Diluted hydrochloric acid was added to an aqueous solution
of carboxymethylcellulose sodium salt ("APP-84" manufactured by
Nippon Paper Chemicals Co., Ltd., a compound having an average
molecular weight of 17,000 and an average of 0.7 carboxyl groups
per one glucose unit) until the solution has a pH of 2 or less. The
resulting solution was dialyzed and concentrated to obtain a 1%
aqueous solution of a carboxymethylcellulose CMC-1.
4-2-2D. Synthesis Example 11
Synthesis of Saccharide CMD-1
[0197] 0.72 g of sodium hydroxide and 1.04 g of bromoacetic acid
were added to 2.5 g of a 10 wt % aqueous solution of Dextran T500
(average molecular weight: 500,000) manufactured by Pharmacia AB,
and the mixture was stirred for several minutes to homogenize. The
solution was maintained at 40.degree. C. for 60 hours and then
cooled with ice. After the addition of diluted hydrochloric acid to
make the pH 2 or less, the solution was dialyzed and freeze-dried
to obtain a carboxymethyldextran CMD-1. Carboxylic acid contained
in CMD-1 was measured by titration to find that CMD-1 contained an
average of 0.4 carboxylic acid groups per one glucose unit.
4-2-3. Experimental Example 8
4-2-3A. Preparation of Carrier Polymer Particles
[0198] Particles isolated from the water dispersion of magnetic
particles A-6 obtained in Synthetic Example 8 by centrifugal
separation were dispersed in acetone. After repeating a procedure
of separating the particles by magnetism and washing five times,
the particles were again dispersed in acetone and the supernatant
liquid was removed by centrifugal separation. The particles
obtained were dried. 0.50 g of the particles was put into a 100 ml
flask and 25 g of ethylenediamine was added. Particles were
irradiated with indirect ultrasonic radiation for 20 minutes and
dispersed. The dispersion liquid was stirred at 50.degree. C. in a
nitrogen atmosphere for six hours, followed by isolation of the
particles by centrifugal separation. The particles were washed five
times with methanol and dried to obtain 0.49 g of aminated
particles Am-6 as a brown powder. Comparison of the infrared
absorption spectrum of the aminated particles Am-6 (after
ethylenediamine treatment) with the infrared absorption spectrum of
the magnetic particles A-6 (before ethylenediamine treatment)
indicates that peaks typical to a primary amine appeared around
3,300 cm.sup.-1 and 3,400 cm.sup.-1 in the infrared absorption
spectrum of the aminated particles Am-6. The aminated particles
Am-6 were thus confirmed to have an amino group introduced into the
magnetic organic polymer particles A-6.
[0199] 150 mg of the aminated particles Am-6 was added to 3.75 g of
a 1% aqueous solution of CMC-1 which was obtained in Synthetic
Example 10. The dispersion liquid was irradiated with indirect
supersonic waves for 20 minutes while cooling with ice. Next, 25.05
mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
was added to the dispersion liquid and the mixture was stirred
under ice cooling for 20 hours. The particles were isolated by
magnetic separation and dispersed in purified water. A procedure of
magnetic separation and washing by dispersing in purified water was
repeated five times. The resulting particles were again dispersed
in purified water and centrifuged to remove the supernatant liquid,
followed by drying to obtain 147 mg of carrier polymer particles
P-9.
4-2-3B. Preparation of Probe-Bonded Polymer Particles
[0200] The carrier polymer particles obtained in Experimental
Example 8 were diluted with and dispersed in purified water to
obtain a water dispersion with a particle concentration of 1 wt %.
500 microliters of the dispersion liquid was put into a
microcentrifuge tube ("Safe-Lock tube" manufactured by Eppendorf)
and magnetically centrifuged using a magnetic stand ("Magical
Trapper" manufactured by Toyobo Co., Ltd.) to remove the
supernatant liquid. After washing three times with a 50 mM
MES--NaOH buffer solution (pH 6, hereinafter referred to as
"Buffer-1"), 0.8 mg of N-hydroxysuccinic acid imide (NHS) and 0.88
mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were
added and stirred. Then, 0.05 mg of a protein
(anti-alpha-fetoprotein antibody) which acts as a probe for
specifically trapping the target material (alpha-fetoprotein,
hereinafter referred to from time to time as "AFP") was added and
the mixture was stirred at room temperature for two hours. After
the reaction, the particles were separated by magnetic separation
and the supernatant liquid was removed. Then, 500 microliters of a
PBS (-) buffer solution containing 1% ethanol amine was added and
the mixture was stirred at room temperature for two hours.
Furthermore, after washing five times with a PBS (-) buffer
solution, the particles were dispersed in 500 microliters of PBS
(-) buffer solution to obtain a dispersion liquid of probe
(antibody)-bonded particles.
4-2-3C. Evaluation of Specific Trapping Property
[0201] The specific trapping property of the probe-bonded particles
obtained in this Experimental Example was evaluated according to
the following method.
4-2-3C-1. Target Material (Protein) Adsorption Reaction Step
[0202] 100 microliters of the above dispersion liquid of the
probe-bonded particles was sampled in a separate tube. Particles
were magnetically separated to remove the supernatant liquid. 500
microliters of a human blood serum solution containing 200 ng/ml of
protein (human alpha-fetoprotein (AFP)) which is the target
material was added to the particles. The mixture was vibrated by a
touch mixer to disperse the particles in the solution, followed by
mixing by rotation and inversion for two hours at room
temperature.
4-2-3C-2. Washing Step
[0203] After magnetic separation, the supernatant liquid was
removed. 1 ml of 10 mM HEPES was added to the tube and the
particles were dispersed using a touch mixer. After further
repeating the same procedure twice, the content was transferred to
a new microcentrifuge tube to perform magnetic separation, and the
supernatant liquid was removed.
4-2-3C-3. Detaching Step
[0204] After the addition of 50 microliters of a 0.5% aqueous
solution of SDS (sodium dodecylsulfate), the mixture was gently
vibrated to disperse the particles. After allowing the mixture to
stand for 10 minutes, magnetic separation was performed and 20
microliters of the supernatant liquid was collected.
4-2-3C-4. Electrophoresis (SDS-PAGE)
[0205] 2-mercaptoethanol was dissolved in a premix sample buffer
solution manufactured by Bio-Rad Laboratories, Inc. to a
concentration of 2 wt % (this solution is hereinafter referred to
as "sample buffer"). 20 microliters of the solution was collected
in a microcentrifuge tube. 20 microliters of the supernatant liquid
collected in the above step was mixed and heated at 100.degree. C.
for five minutes in a tube heater.
[0206] As controls, a 1 mg/ml AFP/BSA (-) solution was diluted with
an SDS solution to 100 fold, 200 fold, and 500 fold. 20 microliters
of each of the diluted solutions were mixed with 20 microliters of
the sample buffer and heated by a block heater at 100.degree. C.
for five minutes. The resulting solutions are called reference AFP
dilutions.
[0207] The reference AFP dilutions were applied to a vertical
electrophoresis system ("Mini-PROTEAN3" manufactured by Bio-Rad
Laboratories, Inc.) in an amount of 20 microliters per one lane to
perform electrophoresis using a precast polyacrylamide gel ("Ready
Gel J" (15%) manufactured by Bio-Rad Laboratories, Inc.) and a
premix electrophoresis buffer solution manufactured by Bio-Rad
Laboratories, Inc. The gel was stained by a standard staining
method using "Silver Stain Plus Kit" manufactured by Bio-Rad
Laboratories, Inc. The stained gel was scanned using a densitometer
"GS-700" manufactured by Bio-Rad Laboratories, Inc. to produce an
image. The product of the concentration and the area of the AFP
band in the gel were determined using an analysis software
"Multi-Analyst".
[0208] Since the weight of AFP which flows per one lane of the gel
is known in the reference dilution AFP, a calibration curve was
drawn from the product of the concentration and the area of the
band, and the amount of AFP detached from the particles was
converted on a weight basis based on the calibration curve. The
resulting weight corresponded to the amount of AFP which had been
adsorbed per 0.2 mg of the particles.
4-2-4. Experimental Example 9
4-2-4A. Preparation of Carrier Polymer Particles
[0209] 0.48 g of aminated particles Am-7 were obtained in the same
manner as in Experimental Example 8, except for using the magnetic
particles A-7. CMD-1 (150 mg) which was obtained in Synthetic
Example 11 was dissolved in 6 g of purified water and 150.5 mg of
the aminated particles Am-7 was added and dispersed in the solution
by irradiation of indirect supersonic waves for 20 minutes. Next,
the dispersion liquid was cooled with ice, and 1.40 g of a 5 wt %
aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride was added. The mixture was stirred under ice cooling
for 12 hours. The particles were isolated by magnetic separation
and dispersed in purified water. A procedure of magnetic separation
and washing by dispersing in purified water was repeated five
times. The resulting particles were again dispersed in purified
water and centrifuged to remove the supernatant liquid, followed by
drying to obtain 147 mg of carrier polymer particles P-10.
4-2-4B. Preparation of Probe-Bonded Polymer Particles and
Evaluation of Specific Trapping Property
[0210] Probe (antibody)-bonded particles were obtained in the same
manner as in Experimental Example 8 by using the carrier polymer
particles P-10 obtained in this Experimental Example. The specific
trapping property of the probe-bonded particles was evaluated
according to the same method as in Experimental Example 8.
4-2-5. Comparative Example 4
[0211] Probe-bonded particles were obtained in the same manner as
in Experimental Example 8 by using magnetic particles "MAG2101"
manufactured by JSR Corporation. The specific trapping property of
the probe-bonded particles was evaluated according to the same
method as in Experimental Example 8. The surface of the magnetic
particles used in the Comparative Example 4 was not covered with a
saccharide.
4-2-6. Evaluation Result of Specific Trapping Property
[0212] FIG. 3 is a photograph showing the evaluation results of the
specific trapping property (an electrophoresis pattern of proteins
adsorbed on the probe-bonded particles) obtained in Experimental
Examples 8 and 9 and Comparative Example 4.
[0213] In FIG. 3, lane 1 indicates proteins trapped by the
probe-bonded particles which were prepared by using the carrier
polymer particles P-9 of Experimental Example 8, lane 2 indicates
proteins trapped by the probe-bonded particles which were prepared
by using the carrier polymer particles P-10 of Experimental Example
9, lane 3 indicates proteins trapped by the probe-bonded particles
which were prepared by using the magnetic particles of Comparative
Example 4, lane 4 indicates the target material (AFP) 20 ng which
is a control, lane 5 indicates the target material (AFP) 50 ng
which is a control, lane 6 indicates the target material (AFP) 100
ng which is a control, and lane 7 indicates a molecular weight
marker.
[0214] It can be understood from FIG. 3 that by using the
probe-bonded particles which were prepared by using the
polymer-bonded particles P-9 of Experimental Example 8, only the
target material (AFP) band was collected from the serum in an
amount of 11 ng per 0.2 mg of the particles. By using the
antibody-bonded particles which were prepared by using the
polymer-bonded particles P-10 of Experimental Example 9, only the
target material (AFP) band was collected from the serum in an
amount of 15 ng per 0.2 mg of the particles. On the other hand,
although a number of bands of blood serum proteins nonspecifically
collected were confirmed in the particles of Comparative Example 4,
it was difficult to confirm the target AFP band.
[0215] The probe-bonded particles of Experimental Examples 8 and 9
were thus confirmed to exhibit only small nonspecific protein
adsorption. Based on these results, it can be understood that since
the surface of the particles is covered with a saccharide and the
probe to specifically trap the target compound chemically bonds to
the saccharide, the probe-bonded particles of Experimental Examples
8 and 9 exhibit only small nonspecific protein adsorption. On the
other hand, the magnetic particles of Comparative Example 4
exhibited large nonspecific adsorption of proteins. It can thus be
understood that the nonspecific adsorption is large if the surface
of the particles is not covered with a saccharide.
4-3. Example 3
4-3-1. Evaluation Method
4-3-1A. Evaluation 1 of Nonspecific Adsorption (Evaluation of
Nonspecific Adsorption of Proteins)
4-3-1A-1. Pre-Washing Step
[0216] The carrier polymer particles prepared in the
later-described Experimental Examples and Comparative Examples were
diluted with and dispersed in purified water to obtain dispersion
liquids, each having a particle concentration of 1 wt %. 500
microliters of the dispersion liquid was put into a microcentrifuge
tube ("Safe-Lock tube" manufactured by Eppendorf) and centrifuged
(15,000 rpm, 15.degree. C., 10 minutes) using a centrifugal
separator ("MX-150" manufactured by Tomy Seiki Co.) to remove the
supernatant liquid. 500 microliters of a PBS(-) buffer solution was
added to the tube which contained a precipitate, and the mixture
was vibrated by a touch mixer to disperse the particles.
4-3-1A-2. Protein Adsorption Reaction Step
[0217] Then, 500 microliters of a PBS(-) solution of 1 wt % BSA
(bovine serum albumin) was added to the tube and the mixture was
vibrated by a touch mixer to disperse the particles in the
solution, followed by mixing by rotation and inversion for two
hours at room temperature.
]
4-3-1A-3. Washing Step
[0218] After centrifugal separation, the supernatant liquid was
removed. 1 ml of 10 mM HEPES was added to the tube and the
particles were dispersed by vibration using a touch mixer. After
further repeating the same procedure twice, the content was
transferred to another microcentrifuge tube to perform centrifugal
separation, and the supernatant liquid was removed.
4-3-1A-4. Detaching Step
[0219] After the addition of 50 microliters of a 0.5% aqueous
solution of SDS (sodium dodecylsulfate), the mixture was gently
vibrated by a touch mixer to disperse the particles. After allowing
the mixture to stand for 10 minutes, the centrifugal separation was
performed and 20 microliters of the supernatant liquid was
collected.
4-3-1A-5. Sampling Step
[0220] 2-mercaptoethanol was dissolved in a premix sample buffer
solution manufactured by Bio-Rad Laboratories, Inc. to a
concentration of 2 wt % (this solution is hereinafter referred to
as "sample buffer"). 20 microliters of the solution was collected
in the microcentrifuge tube. 20 microliters of the supernatant
liquid collected in the above step was mixed and heated at
100.degree. C. for five minutes in a tube heater.
[0221] As controls, a 1 wt % BSA solution in PBS(-) was diluted
with 2% SDS to 5,000 fold, 10,000 fold, and 20,000 fold. 20
microliters of each of the diluted solutions was mixed with 20
microliters of the sample buffer and heated in a tube heated at
100.degree. C. for five minutes. The resulting solutions are called
reference BSA dilutions.
4-3-1A-6. Electrophoresis (SDS-PAGE)
[0222] The reference AFP dilutions were applied to a vertical
electrophoresis system ("Mini-PROTEAN3" manufactured by Bio-Rad
Laboratories, Inc.) in an amount of 20 microliters per one lane to
perform electrophoresis using a precast polyacrylamide gel ("Ready
Gel J" (15%) manufactured by Bio-Rad Laboratories, Inc.) and a
premix electrophoresis buffer solution manufactured by Bio-Rad
Laboratories, Inc. The gel was stained by a standard staining
method using "Silver Stain Plus Kit" manufactured by Bio-Rad
Laboratories, Inc. The stained gel was scanned using a densitometer
"GS-700" manufactured by Bio-Rad Laboratories, Inc. to produce an
image. The product of the concentration and the area of the BSA
band in the gel were determined using an analysis software
"Multi-Analyst".
[0223] Since the weight of BSA which flows per one lane of the gel
is known in the dilution BSA for reference, a calibration curve was
drawn from the product of the band concentration and the area, and
the amount of BSA detached from the particles was converted on a
weight basis based on the calibration curve. The resulting weight
corresponded to the amount of BSA which had been adsorbed per one
mg of the particles.
4-3-1B. Evaluation of Specific Trapping Property
4-3-1B-1. Preparation of Probe-Bonded Polymer Particles
[0224] Carrier polymer particles prepared in the later-described
Experimental Examples and Comparative Examples were diluted with
and dispersed in purified water to obtain dispersion liquids, each
having a particle concentration of 1 wt %. 500 microliters of the
water dispersion was put into a microcentrifuge tube and
centrifuged to remove the supernatant liquid. After washing three
times with a 50 mM MES--NaOH buffer solution (pH 6, hereinafter
referred to as "Buffer-1"), 0.8 mg of N-hydroxysuccinic acid imide
(NHS) and 0.88 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) were added and stirred. Then, 0.05 mg of a protein
(anti-alpha-fetoprotein antibody) which acts as a probe for
specifically catching the target material (alpha-fetoprotein,
hereinafter referred to from time to time as "AFP") was added and
the mixture was stirred at room temperature for two hours. After
the reaction, the particles were separated by centrifugal
separation and the supernatant liquid was removed. Then, 500
microliters of a PBS (-) buffer solution containing 1% ethanol
amine was added and the mixture was stirred at room temperature for
two hours. Furthermore, after washing five times with a PBS (-)
buffer solution, the particles were dispersed in 500 microliters of
PBS (-) buffer solution to obtain a dispersion liquid of probe
(antibody)-bonded particles.
4-3-1B-2. Target Material (Protein) Adsorption Reaction Step
[0225] 100 microliters of the above dispersion liquid of the
probe-bonded particles was sampled in a separate tube. The
particles were separated by centrifugation to remove the
supernatant liquid. 500 microliters of a human blood serum solution
containing the target protein (human alpha-fetoprotein (AFP)) which
is the target material was added to the particles. The mixture was
vibrated by a touch mixer to disperse the particles in the
solution, followed by mixing by rotation and inversion for two
hours at room temperature.
4-3-1B-3. Washing Step
[0226] After centrifugal separation, the supernatant liquid was
removed. 1 ml of 10 mM HEPES was added to the tube and the
particles were dispersed using a touch mixer. After further
repeating the same procedure twice, the content was transferred to
another microcentrifuge tube to perform centrifugal separation, and
the supernatant liquid was removed.
4-3-1B-4. Detaching Step
[0227] After the addition of 50 microliters of a 0.5% aqueous
solution of SDS (sodium dodecylsulfate), the mixture was gently
vibrated by a touch mixer to disperse the particles. After allowing
the mixture to stand for 10 minutes, the centrifugal separation was
performed and 20 microliters of a supernatant liquid was
collected.
4-3-1B-5. Sampling Step
[0228] 2-mercaptoethanol was dissolved in a premix sample buffer
solution manufactured by Bio-Rad Laboratories, Inc. to a
concentration of 2 wt % (this solution is hereinafter referred to
as "sample buffer"). 20 microliters of the solution was collected
in the microcentrifuge tube. 20 microliters of the supernatant
liquid collected in the above step was mixed and heated at
100.degree. C. for five minutes in a tube heater.
[0229] As controls, a 1 mg/ml AFP/PBS (-) solution was diluted with
an SDS solution to 100 fold, 200 fold, and 500 fold. 20 microliters
of each of the diluted solutions was mixed with 20 microliters of
the sample buffer and heated by a block heater at 100.degree. C.
for five minutes. The resulting solutions are called reference AFP
dilutions.
4-3-1B-6. Electrophoresis (SDS-PAGE)
[0230] The electrophoresis was carried out in the same manner as in
4.1.1F except for using AFP instead of BSA.
[0231] Since the weight of AFP which flows per one lane of the gel
is known in the reference dilution AFP, a calibration curve was
drawn from the product of the concentration and the area of the
band, and the amount of AFP detached from the particles was
converted on a weight basis based on the calibration curve. The
resulting weight corresponded to the amount of AFP which had been
adsorbed per 0.2 mg of the particles.
4-3-1C. Particle Diameter
[0232] The diameter of the particles with a diameter of 1
micrometer or more was measured using a laser diffraction particle
size distribution analyzer ("SALD-200V" manufactured by Shimadzu
Corp.) and the diameter of the particles with a diameter of less
than 1 micrometer was measured using a particle size distribution
analyzer based on a laser dispersion diffraction method ("LS 13
320" manufactured by Beckmann Coulter).
4-3-1D Infrared Absorption Spectrum
[0233] The infrared absorption spectrum was measured by a KBr
method using a Fourier-transform infrared spectrophotometer
("JIR-5500" manufactured by JEOL Ltd.).
4-3-2. Synthesis Examples
4-3-2A. Synthesis Example 12
Synthesis of Organic Polymer Particles A-8
[0234] The organic polymer particles A-8 were prepared by a
two-step swelling polymerization method using seed particles.
[0235] Using polystyrene particles with a particle diameter of 0.98
micrometers obtained by soap-free polymerization as seed particles,
a water dispersion (solid content: 5.0 g) was prepared by
dispersing these polystyrene particles in 500 g of water in a
nitrogen atmosphere. According to the two step swelling
polymerization method (based on the method described in
JP-B-57-24369), an organic solvent (0.1 g of "Shellsol TK") was
added to the seed particles as a first step, and monomers (10 g of
TMP (trimethylolpropane trimethacrylate) and 90 g of GMA (glycidyl
methacrylate)) were added as a second step to cause them to be
adsorbed. Then, 2 g of AIBN (azobisisobutyronitrile) was added and
the mixture was slowly stirred at 75.degree. C. for 24 hours. The
reaction solution was cooled and filtered through a 500 mesh wire
gauze to confirm that 99% of the product passed through the wire
gauze. The polymerization stability was good. The polymerization
yield was 99%. The particle diameter of the resulting organic
polymer particles A-8 was 2.58 micrometers, the coefficient of
variation of the particle diameter was 2.3%, and the particles were
monodisperse particles.
4-3-2B. Synthesis Example 13
Synthesis of Organic Polymer Particles A-9
[0236] Organic polymer particles A-9 with a particle diameter of
2.61 micrometers and a coefficient of variation of 2.1% were
obtained in the same manner as in Synthetic Example 12, except for
using 30 g of MMA, 10 g of TMP, and 60 g of GMA as monomers.
4-3-2C. Synthesis Example 14
Synthesis of Saccharide CMC-1
[0237] Diluted hydrochloric acid was added to an aqueous solution
of carboxymethylcellulose sodium salt ("APP-84" manufactured by
Nippon Paper Chemicals Co., Ltd., a compound having an average
molecular weight of 17,000 and an average of 0.7 carboxyl groups
per one glucose unit) until the solution has a pH of 2 or less. The
resulting solution was dialyzed and concentrated to obtain a 1%
aqueous solution of carboxymethylcellulose CMC-1.
4-3-3. Experimental Example 10
4-3-3A. Preparation of Carrier Polymer Particles P-11
[0238] The polymer particles isolated from the dispersion liquid of
organic polymer particles A-8 by centrifugation were washed by
dispersing in acetone, followed by centrifugation. This washing
procedure was repeated three times. The resulting particles were
dried. 0.50 g of the particles was put into a 200 ml flask and 5 g
of acetone and 75 g of 1% sulfuric acid were added. Particles were
irradiated with indirect ultrasonic radiation for 20 minutes and
dispersed. The dispersion liquid was heated at 60.degree. C. for
two hours while stirring, followed by isolation of the particles by
centrifugal separation. The particles were washed three times with
water and dried to obtain 0.51 g of organic polymer particles Hy-1
as a white powder.
[0239] The weight of the organic polymer particles Hy-1 was larger
than the weight of the organic polymer particles A-8. Comparison of
the infrared absorption spectrum of the organic polymer particles
Hy-1 (after sulfuric acid treatment) with the infrared absorption
spectrum of the organic polymer particles A-8 (before sulfuric acid
treatment) indicates that in the infrared absorption spectrum of
the organic polymer particles Hy-1, a peak originating from an
epoxy group, which was observed around 900 cm.sup.-1 of the
infrared absorption spectrum of the organic polymer particles A-8,
was weak and, instead, a broad peak due to a hydroxyl group was
observed around 3,500 cm.sup.-1. Based on the above results, the
organic polymer particles Hy-1 were confirmed to have been obtained
by a partial hydrolysis of epoxy groups in the organic polymer
particles A-8 and introduction of hydroxyl groups.
[0240] 0.50 g of the organic polymer particles Hy-1 was put into a
100 ml flask and 25 g of ethylenediamine was added. Particles were
irradiated with indirect ultrasonic radiation for 10 minutes and
dispersed. The dispersion liquid was stirred at 50.degree. C. in a
nitrogen atmosphere for six hours, followed by isolation of the
particles by centrifugal separation. The particles were washed four
times with methanol and dried to obtain 0.61 g of organic polymer
particles Am-8 as a white powder.
[0241] The weight of the organic polymer particles Am-8 was larger
than the weight of the organic polymer particles Hy-1. Comparison
of the infrared absorption spectrum of the organic polymer
particles Am-8 (after ethylenediamine treatment) with the infrared
absorption spectrum of the organic polymer particles Hy-1 (before
ethylenediamine treatment) indicates that in the infrared
absorption spectrum of the organic polymer particles Am-8, a peak
originating from an epoxy group, which was observed around 900
cm.sup.-1 of the infrared absorption spectrum of the organic
polymer particles Hy-1, disappeared and, instead, peaks typical to
primary amine appeared around 3,300 cm.sup.-1 and 3,500 cm.sup.-1.
Based on the results, the organic polymer particles Am-8 were
confirmed to have an amino group introduced into the organic
polymer particles Hy-1.
[0242] 6 mg of N-hydroxysuccinic acid imide was added to 3 g of a
1% aqueous solution of CMC-1 which was obtained in Synthetic
Example 14, and the mixture was stirred at room temperature for 10
minutes. Next, the solution was cooled with ice and 20 mg of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was
added. The mixture was stirred under ice cooling for 30 minutes.
Next, 30.1 mg of the above organic polymer particles Am-8 was added
to the solution. The mixture was irradiated with indirect
ultrasonic radiation for 20 minutes and stirred under ice cooling
for 12 hours. The particles were isolated by centrifugal
separation, dispersed in purified water, isolated by centrifugal
separation, and washed. This procedure was repeated 10 times,
followed by drying to obtain 35.0 mg of carrier polymer particle
precursor pre-P-11.
[0243] In addition to the peaks originating from the organic
polymer particles Am-8 before the reaction, peaks originating from
carboxymethylcellulose were observed around 3,400 cm.sup.-1 and
1,600 cm.sup.-1 in the infrared absorption spectrum of the carrier
polymer particle precursor pre-P-11. Based on these results, the
carrier polymer particle precursor pre-P-11 was confirmed to have a
saccharide (carboxymethylcellulose) bonded to the organic polymer
particles Am-8.
[0244] An operation of dispersing the carrier polymer particle
precursor pre-P-11 (20.2 mg) in 2 ml of a 0.01 M sodium hydroxide
aqueous solution and isolating the particles by centrifugation was
carried out three times, then an operation of dispersing the
particles in purified water and isolating by centrifugal separation
was carried out three times, followed by drying to obtain 18.6 mg
of carrier polymer particles P-11.
[0245] The peaks originating from carboxymethylcellulose were still
observed around 3,400 cm.sup.-1 and 1,600 cm.sup.-1 in the infrared
absorption spectrum of the carrier polymer particles P-11, although
these peaks were weaker as compared with those in the carrier
polymer particle precursor pre-P-11 before the reaction. The
carrier polymer particles P-11 were thus confirmed to have a
saccharide (carboxymethylcellulose) bonded to the organic polymer
particles Am-8. The carrier polymer particles P-11 were further
treated with a 0.01 M aqueous solution of sodium hydroxide to find
that the weight loss was less than 0.1 mg, which is a negligible
amount.
4-3-3B. Evaluation Result of Nonspecific Absorption of Protein
[0246] The nonspecific protein adsorption of the carrier polymer
particles P-11 was measured according the above-described method to
confirm that the value was less than the detectable limit (0.01
ng/mg).
4-3-4. Experimental Example 11
4-3-4A. Preparation of Carrier Polymer Particles P-12
[0247] The polymer particles isolated from the dispersion liquid of
organic polymer particles A-9 by centrifugation were washed by
dispersing in acetone, followed by centrifugation. This washing
procedure was repeated three times. The resulting particles were
dried. 0.50 g of the particles were put into a 100 ml flask and 25
g of ethylenediamine was added. Particles were irradiated with
indirect ultrasonic radiation for 10 minutes and dispersed. The
dispersion liquid was stirred at 50.degree. C. in a nitrogen
atmosphere for six hours, followed by isolation of the particles by
centrifugal separation. The particles were washed four times with
methanol and dried to obtain 0.61 g of organic polymer particles
Am-9 as a white powder. 29.9 mg of the organic polymer particles
Am-9 was added to 3 g of a 1% aqueous solution of CMC-1 which was
obtained in Synthetic Example 14. The mixture was irradiated with
indirect supersonic wave for 20 minutes to disperse the particles.
Next, the solution was cooled with ice and 20 mg of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was
added. The mixture was stirred under ice cooling for 12 hours. The
procedure of isolation and drying was carried out in the same
manner as in Experimental Example 10 to obtain 36.2 g of carrier
polymer particle precursor pre-P-12. Next, the procedure of
Experimental Example 10 was followed, except for using carrier
polymer particle precursor pre-P-12 (20.3 mg) instead of the
carrier polymer particle precursor pre-P-11 of the Experimental
Example 10, to obtain 17.9 mg of carrier polymer particle precursor
pre-P-12.
4-3-4B. Evaluation of Nonspecific Adsorption
[0248] The nonspecific protein adsorption of the carrier polymer
particles P-12 was measured according the above-described method to
confirm that the value was very low (0.02 ng/mg).
4-3-5. Comparative Example 5
[0249] The nonspecific protein adsorption of the organic polymer
particles A-8 was measured according the above-described method to
confirm that the value was high (1.1 ng/mg).
4-3-6. Comparative Example 6
[0250] Commercially available standard polystyrene particles
("STADEX SC200S" manufactured by JSR Corporation) was sufficiently
washed with purified water and their nonspecific protein adsorption
was measured according the above-described method to confirm that
the value was very high (20 ng/mg).
4-3-7. Comparative Example 7
[0251] The nonspecific protein adsorption of the carrier polymer
particle precursor pre-P-11 was measured according the
above-described method to confirm that the value was very low (0.02
ng/mg), but not lower than that of the carrier polymer particles
P-11, of which the nonspecific protein adsorption was less than the
detectable limit.
4-3-8. Evaluation Result of Specific Trapping Property
[0252] FIG. 4 is a photograph showing the evaluation results of the
specific trapping property (an electrophoresis pattern of proteins
adsorbed on the probe-bonded particles) of carrier polymer
particles P-11 and P-12 obtained respectively in Experimental
Examples 10 and 11, and the carrier polymer particle precursor
pre-P-11 obtained in Comparative Example 7.
[0253] In FIG. 4, lane 1 indicates proteins trapped by the
probe-bonded particles which were prepared by using the carrier
polymer particles P-11 of Experimental Example 10, lane 2 indicates
proteins trapped by the probe-bonded particles which were prepared
by using the carrier polymer particles P-12 of Experimental Example
11, lane 3 indicates proteins trapped by the probe-bonded particles
which were prepared by using the carrier polymer particle precursor
pre-P-11 of Comparative Example 7, lane 4 indicates the target
material (AFP) 20 ng which is a control, lane 5 indicates the
target material (AFP) 50 ng which is a control, and lane 6
indicates a molecular weight marker.
[0254] It can be understood from FIG. 4 that using the probe-bonded
particles which were prepared by using the carrier polymer
particles P-11 of Experimental Example 10, only the target material
(AFP) band was collected from the serum in an amount of 16 ng per
0.2 mg of the particles. By using the antigen-bonded particles
which were prepared by using the polymer-bonded particles P-12 of
Experimental Example 11, only the target material (AFP) band was
collected from the serum in an amount of 12 ng per 0.2 mg of the
particles. On the other hand, it was difficult to confirm the
target AFP band in the particles of Comparative Example 7.
[0255] As a result of the above experiments, the probe-bonded
particles formed using the carrier polymer particles of
Experimental Examples 10 and 11 were confirmed to exhibit only
small nonspecific protein adsorption and to be able to specifically
trap the target material. On the other hand, the magnetic particles
of Comparative Example 7 could not specifically trap a target
material, although the particles exhibited small nonspecific
protein adsorption. It can thus be understood that the particles
with a physically-adsorbed saccharide on the surface covered with a
saccharide, such as in the particles of Comparative Example 7,
cannot exhibit sufficient nonspecific trapping performance of a
target material.
* * * * *