U.S. patent application number 13/152802 was filed with the patent office on 2011-09-29 for organic polymer particles and process for producing same.
This patent application is currently assigned to JSR CORPORATION. Invention is credited to Takahiro KAWAI, Eiji TAKAMOTO, Kouji TAMORI.
Application Number | 20110233454 13/152802 |
Document ID | / |
Family ID | 39260235 |
Filed Date | 2011-09-29 |
United States Patent
Application |
20110233454 |
Kind Code |
A1 |
TAMORI; Kouji ; et
al. |
September 29, 2011 |
ORGANIC POLYMER PARTICLES AND PROCESS FOR PRODUCING SAME
Abstract
Organic polymer particles having a structure shown by the
following formula (1) are disclosed. ##STR00001## wherein A
represents an alkylidene group, an alkylene group, a cyclohexylene
group, or a phenylene group, and B represents a linear or branched
alkylene group or an alkylidene group having 1 to 6 carbon
atoms.
Inventors: |
TAMORI; Kouji;
(Tsuchiura-shi, JP) ; TAKAMOTO; Eiji;
(Tsuchiura-shi, JP) ; KAWAI; Takahiro;
(Tsuchiura-shi, JP) |
Assignee: |
JSR CORPORATION
Chuo-ku
JP
|
Family ID: |
39260235 |
Appl. No.: |
13/152802 |
Filed: |
June 3, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11773801 |
Jul 5, 2007 |
7981512 |
|
|
13152802 |
|
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Current U.S.
Class: |
252/62.54 ;
526/318 |
Current CPC
Class: |
C08G 63/181 20130101;
C08G 63/199 20130101; C08G 63/52 20130101; Y10T 428/2991 20150115;
Y10T 428/2998 20150115 |
Class at
Publication: |
252/62.54 ;
526/318 |
International
Class: |
C08F 222/16 20060101
C08F222/16; H01F 1/36 20060101 H01F001/36 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 28, 2006 |
JP |
2006-264646 |
Claims
1-4. (canceled)
5. Organic polymer particles comprising a structure shown by the
following formula (3): ##STR00008## wherein E represents an
alkylene group or an alkylidene group having 1 to 12 carbon atoms
which may be branched, or a single bond.
6. The organic polymer particles according to claim 5, wherein the
structure represented by the formula (3) is derived from at least
one compound selected from 2-(meth)acryloyloxyethyl phthalate,
2-(meth)acryloyloxypropyl phthalate, 20 p-vinylbenzoic acid, and
vinylphenylacetic acid.
7-8. (canceled)
9. The organic polymer particles according to claim 5, comprising a
magnetic material.
10-11. (canceled)
12. The organic polymer particles according to claim 5, wherein the
particle surface comprises a copolymer made from a compound (A)
having a structure of the formula (3) and a polymerizable
unsaturated group, and another copolymerizable monomer (B).
13-14. (canceled)
15. The organic polymer particles according to claim 12, comprising
mother particles containing nuclear particles and a magnetic
material layer of superparamagnetic fine particles formed on the
surface of the nuclear particles, and a copolymer layer made from
the copolymer which is formed to cover the mother particles.
16-17. (canceled)
18. The organic polymer particles according to claim 5 used for
protein bonding.
19-20. (canceled)
21. A process for producing organic polymer particles comprising
polymerizing a monomer part comprising a compound having a
structure of the above formula (3) and an ethylenically unsaturated
group.
Description
[0001] Japanese Patent Application No. 2006-264646 filed on Sep.
28, 2006 is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to organic polymer particles
that can chemically bond to biological-related substances,
including proteins such as an antibody or an antigen, by utilizing
a carboxyl group, and can exhibit high detection sensitivity, and
to a process for producing the same.
[0003] Organic polymer particles and magnetic particles are used as
a reaction solid phase of a diagnostic agent using an
antigen-antibody reaction in order to detect substances to be
examined such as infections, cancer markers, and hormones, for
example. In such a diagnostic agent, a probe (primary probe) for
inspecting an antibody or an antigen is immobilized on the
particles. A substance to be inspected in a sample reacts with a
second inspection probe after having been trapped on the particles
via the primary probe. The second inspection probe (secondary
probe) is labeled with a fluorescent substance or an enzyme,
whereby the target substance is detected by fluorescence or by an
enzyme reaction.
[0004] In recent years, due to a demand for an increase in the
inspection sensitivity for the early detection of diseases, an
increase in sensitivity of a diagnostic agent has been an important
subject. In order to increase sensitivity of diagnostic agents
using magnetic particles, a method of using enzyme coloring as a
detecting means is being replaced by a method of using fluorescence
or chemiluminescence, both of which ensure higher sensitivity.
[0005] Development of these detection techniques are said to have
reached a level in which a one molecule-substance for inspection
can be theoretically detected. In practice, however, sensitivity is
still insufficient. The incapability of maintaining the activity of
the primary probe after bonding due to change in the conformation
of the protein, which is the primary probe bonded to the particles,
can be given as a reason for this.
[0006] Generally, as methods for maintaining the activity of such a
primary probe, a method of causing a polyhydric alcohol to be
present when the primary probe is bonded, and a method of bonding
the primary probe via a hybrid protein bonded to the particles are
disclosed (JP-A-9-304386 and WO 97/35964). However, the activity of
the primary probe is insufficient when the primary probe is bonded
by the method of causing a polyhydric alcohol to be present
together. The method of using a hybrid protein involves a
complicated and high-cost production process.
SUMMARY
[0007] An object of the invention is to provide organic polymer
particles that can chemically bond to biological-related
substances, including proteins such as an antibody or an antigen,
by utilizing a carboxyl group, and can exhibit high detection
sensitivity, and to a process for producing the same.
[0008] In order to achieve the above object, the inventors have
conducted extensive studies and found that organic polymer
particles made from a polymer containing a carboxylic acid monomer
scarcely soluble in water exhibit outstandingly high sensitivity in
the fields of biological chemistry and medical supplies. This
finding has led to the completion of the invention.
[0009] Organic polymer particles according to one aspect of the
invention have a structure shown by the following formula (1).
##STR00002##
wherein A represents an alkylidene group, an alkylene group, a
cyclohexylene group, or a phenylene group, and B represents a
linear or branched alkylene group or an alkylidene group having 1
to 6 carbon atoms.
[0010] The structure represented by the formula (1) may be derived
from at least one compound selected from 2-(meth)acryloyloxyethyl
succinate, 2-(meth)acryloyloxyethyl phthalate,
2-(meth)acryloyloxyethyl hexahydrophthalate,
2-(meth)acryloyloxypropyl succinate, 2-(meth)acryloyloxypropyl
phthalate, and 2-(meth)acryloyloxypropyl hexahydrophthalate.
[0011] Organic polymer particles according to one aspect of the
invention have a structure shown by the following formula (2).
##STR00003##
wherein D represents a linear or branched alkylene group having 2
to 13 carbon atoms.
[0012] The structure represented by the formula (2) may be derived
from at least one compound selected from 2-(meth)acryloyloxyethyl
succinate, 2-(meth) acryloyloxypropyl succinate, myristoleic acid,
palmitoleic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic
acid, erucic acid, nervonic acid, linolic acid, alpha-linolenic
acid, eleostearic acid, stearidonic acid, arachidonic acid,
eicosapentaenoic acid, clupanodonic acid, and docosahexaenoic
acid.
[0013] Organic polymer particles according to one aspect of the
invention have a structure shown by the following formula (3).
##STR00004##
wherein E represents an alkylene group or an alkylidene group
having 1 to 12 carbon atoms which may be branched, or a single
bond.
[0014] The structure represented by the formula (3) may be derived
from at least one compound selected from 2-(meth)acryloyloxyethyl
phthalate, 2-(meth)acryloyloxypropyl phthalate, p-vinylbenzoic
acid, and vinylphenylacetic acid.
[0015] The above organic polymer particles may contain a magnetic
material.
[0016] In the above organic polymer particles, the particle surface
may comprise a copolymer made from a compound (A) having a
structure of any one of the above formulas (1) to (3) and a
polymerizable unsaturated group, and another copolymerizable
monomer (B).
[0017] In this case, the organic polymer particles may comprise
mother particles containing nuclear particles and a magnetic
material layer of superparamagnetic fine particles formed on the
surface of the nuclear particles, and a copolymer layer made from
the above copolymer which is formed to cover the mother
particles.
[0018] The above organic polymer particles may be used for protein
bonding.
[0019] A process for producing organic polymer particles according
to one aspect of the invention comprises polymerizing a monomer
part comprising a compound having a structure of any one of the
above formulas (1) to (3) and an ethylenically unsaturated
group.
[0020] The above organic polymer particles can chemically bond to
biological-related substances, including proteins such as an
antibody or an antigen, by utilizing the carboxyl group, and can
exhibit high detection sensitivity.
[0021] Since the above organic polymer particles have a high
primary probe activity when bonded to a primary probe, the organic
polymer particles can exhibit high sensitivity when applied to a
diagnostic agent and the like. Thus, the organic polymer particles
are useful as a diagnostic agent and the like.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0022] Organic polymer particles of one embodiment of the invention
and a process for producing the organic polymer particles will be
described below.
1. ORGANIC POLYMER PARTICLES AND PROCESS FOR PRODUCING THE SAME
1.1. Constitution of Organic Polymer Particles
[0023] The organic polymer particles according to one embodiment of
the invention have a carboxyl group originating from a compound (A)
possessing a specific structure having a carboxyl group and a
polymerizable unsaturated group (hereinafter referred to from time
to time as "carboxylic acid monomer (A)"). The carboxyl group
originating from the compound (A) refers to the carboxyl group
contained in the carboxylic acid monomer (A).
[0024] The organic polymer particles according to one embodiment of
the invention may have a carboxyl group originating from the
carboxylic acid compound (A) having solubility of 20% by mass or
less in water at 25.degree. C. The carboxylic acid compound (A) may
contain a radically polymerizable unsaturated double bond and a
carboxyl group in one molecule.
[0025] At least the surface of the organic polymer particles
according to this embodiment comprises a polymer part, and the
polymer part may have a carboxyl group originating from the
carboxylic acid compound (A). The polymer part may be either a
polymer formed by polymerization of the carboxylic acid monomer (A)
or a copolymer formed by copolymerization of a monomer part
containing the carboxylic acid monomer (A) and another
copolymerizable monomer (B).
[0026] Either the entirety of the organic polymer particles
according to this embodiment may consist of a polymer part or the
organic polymer particles may have a core-shell structure, with the
shell being formed of a polymer part.
[0027] For example, when the polymer part is prepared by
polymerizing a commonly-used carboxylic acid monomer with high
solubility in water, such as acrylic acid or methacrylic acid, a
water-soluble polymer is easily produced. If such a water-soluble
polymer bonds to the surface of particles, it is thought that
active sites of a biological-related substance (for example, a
protein) bonded to the particle surface is sterically hindered, or
the conformation of the bonded biological-related substance (for
example, a protein) is destroyed by polycarboxylic acid charges,
thereby lowering the activity. As a result, it is thought that the
sensitivity is reduced.
[0028] On the other hand, according to the organic polymer
particles according to this embodiment, since the polymer part is
formed by using a specific carboxylic acid monomer, it is difficult
to produce a water-soluble polymer. It is presumed that this is the
reason why organic polymer particles exhibit high sensitivity.
[0029] In the organic polymer particles according to this
embodiment, the carboxyl group originating from the carboxylic acid
monomer (A) is a factor for exhibiting high sensitivity and
accelerating bonding with a probe for inspection (a primary probe,
for example, a protein) by known activation by means of
esterification or amidation using a water-soluble carbodiimide and
the like. Proteins usable as the primary probe and the method for
bonding will be described later.
[0030] The carboxylic acid monomer (A) has solubility in water at
25.degree. C. of 20% by mass or less, preferably 10% by mass or
less, and most preferably 5% by mass or less. If the solubility in
water at 25.degree. C. of the carboxylic acid monomer (A) is more
than 20% by mass, high sensitivity is not exhibited.
[0031] In the invention, the solubility in water at 25.degree. C.
of the monomer is determined by slowly adding the monomer in
question to distilled water at 25.degree. C. while stiffing until
the monomer separates from the water. The amount (% by mass) of the
monomer in the solution immediately before separation is defined as
the solubility.
[0032] As examples of the carboxylic acid monomer (A),
(meth)acrylic acid derivatives such as 2-(meth)acryloyloxyethyl
succinate, 2-(meth)acryloyloxyethyl phthalate,
2-(meth)acryloyloxyethyl hexahydrophthalate,
2-(meth)acryloyloxypropyl succinate, 2-(meth)acryloyloxypropyl
phthalate, and 2-(meth)acryloyloxypropyl hexahydrophthalate;
aromatic derivatives such as p-vinylbenzoic acid and
vinylphenylacetic acid; and unsaturated fatty acids such as
myristoleic acid, palmitoleic acid, oleic acid, elaidic acid,
vaccenic acid, gadoleic acid, erucic acid, nervonic acid, linolic
acid, alpha-linolenic acid, eleostearic acid, stearidonic acid,
arachidonic acid, eicosapentaenoic acid, clupanodonic acid, and
docosahexaenoic acid can be given. Among the above compounds, in
view of high sensitivity of the resulting organic polymer particles
and ease of polymerization, the carboxylic acid monomer (A) is
preferably a (meth)acrylic acid derivative, more preferably a
monoester compound of a hydroxyalkyl (meth)acrylate and a
dicarboxylic acid, and still more preferably
2-(meth)acryloyloxyethyl succinate, 2-(meth) acryloyloxyethyl
phthalate, or 2-(meth)acryloyloxyethyl hexahydrophthalate, and most
preferably 2-methacryloyloxyethylphthalate.
[0033] The organic polymer particles according to this embodiment
may have the structure shown by the following formulas (1) to
(3).
##STR00005##
wherein A represents an alkylidene group, an alkylene group, a
cyclohexylene group, or a phenylene group, and B represents a
linear or branched alkylene group or an alkylidene group having 1
to 6 carbon atoms.
##STR00006##
wherein D represents a linear or branched alkylene group having 2
to 13 carbon atoms.
##STR00007##
wherein E represents an alkylene group or an alkylidene group
having 1 to 12 carbon atoms which may be branched, or a single
bond.
[0034] In formula (1), as examples of the alkylidene group
represented by A or B, an ethylidene group, an isopropylidene
group, and an isobutylidene group can be given; as examples of the
alkylene group represented by A or B, a methylene group, a
dimethylene group, a trimethylene group, a tetramethylene group, a
pentamethylene group, and a hexamethylene group can be given; as
examples of the cyclohexylene group represented by A, a
1,2-cyclohexylene group, a 1,3-cyclohexylene group, and a
1,4-cyclohexylene group can be given; and as examples of the
phenylene group represented by A, a 1,2-phenylene group, a
1,3-phenylene group, and a 1,4-phenylene group can be given. Among
these, the structure of formula (1) in which the alkylene group
represented by B is a dimethylene group or a trimethylene group is
preferable.
[0035] In formula (2), as examples of the alkylene represented by
D, a dimethylene group, a trimethylene group, a tetramethylene
group, a pentamethylene group, a hexamethylene group, a
heptamethylene group, a nonamethylene group, an undecamethylene
group, and a tridecamethylene group can be given.
[0036] In formula (3), as examples of the alkylidene group
represented by E, an ethylidene group, an isopropylidene group, and
an iso-butylidene group can be given; and as examples of the
alkylene group, a methylene group, a dimethylene group, a
trimethylene group, a tetramethylene group, a pentamethylene group,
and a hexamethylene group can be given.
[0037] The structure shown by the above formulas (1) to (3) may be
included in the above carboxylic acid monomer (A).
[0038] The structure represented by formula (1) is preferably
derived from one of the compounds selected from the group
consisting of 2-(meth)acryloyloxyethyl succinate,
2-(meth)acryloyloxyethyl phthalate, 2-(meth)acryloyloxyethyl
hexahydrophthalate, 2-(meth)acryloyloxypropyl succinate,
2-(meth)acryloyloxypropyl phthalate, 2-(meth)acryloyloxypropyl
hexahydrophthalate, 2-(meth)acryloyloxyethyl succinate,
2-(meth)acryloyloxyethyl phthalate, 2-(meth)acryloyloxyethyl
hexahydrophthalate, 2-(meth)acryloyloxypropyl succinate,
2-(meth)acryloyloxypropyl phthalate, and 2-(meth)acryloyloxypropyl
hexahydrophthalate.
[0039] In addition, for example, the structure represented by
formula (2) is preferably derived from one of the compounds
selected from the group consisting of 2-(meth) acryloyloxyethyl
succinate, 2-(meth)acryloyloxypropyl succinate, myristoleic acid,
palmitoleic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic
acid, erucic acid, nervonic acid, linolic acid, alpha-linolenic
acid, eleostearic acid, stearidonic acid, arachidonic acid,
eicosapentaenoic acid, clupanodonic acid, and docosahexaenoic
acid.
[0040] Furthermore, for example, the structure represented by
formula (3) is preferably derived from one of the compounds
selected from the group consisting of 2-(meth)acryloyloxyethyl
phthalate, 2-(meth)acryloyloxypropyl phthalate, p-vinylbenzoic
acid, and vinylphenylacetic acid.
[0041] Since the structures shown by the above-mentioned formulas
(1) to (3) neither three-dimensionally hinder the active sites of
bonded biological-related substances (for example, a protein) nor
destroy the conformation of bonded biological-related substances
(for example, a protein) on the surface of polymer particles, these
structures enable the polymer particles to exhibit high
sensitivity.
[0042] The organic polymer particles according to this embodiment
may have at least one of the structures shown by the above formulas
(1) to (3).
[0043] The organic polymer particles according to this embodiment
may have a structure shown by the above formulas (1) to (3) at
least on the surface, and it is preferable that the above polymer
part has the structure.
[0044] In the polymer part of the organic polymer particles
according to this embodiment, the amount of the carboxyl group per
the amount of solid components of the particles is preferably from
1 to 300 micromol/g, more preferably from 2 to 200 micromol/g, and
most preferably from 5 to 100 micromol/g. If the amount of the
carboxyl group is less than 1 micromol/g, bonding of a primary
probe may be difficult; on the other hand, if more than 300
micromol/g, non-specific adsorption may increase. The hydrogen ion
of the carboxyl group may be replaced by a cation such as a sodium
ion, a potassium ion, or an ammonium ion.
[0045] The number average particle size (hereinafter referred to
simply as "particle size") of the organic polymer particles
according to this embodiment is preferably from 0.01 to 15
micrometers, more preferably from 0.03 to 10 micrometers, and most
preferably from 0.05 to 10 micrometers. The particle size can be
determined by a laser diffraction-scattering method. If the
particle size is less than 0.01 micrometers, it takes a long time
for separation using centrifugation and the like, resulting in
insufficient separation of the particles from a washing solvent
such as water. This makes it difficult to sufficiently remove
molecules other than target molecules (e.g. biological-related
substances such as proteins and nucleic acids), giving rise to
possible inadequate purification. On the other hand, if the
particle size is more than 15 micrometers, the sensitivity may be
impaired as a result of a decrease in the amount of captured
physiologically active substances due to a small specific surface
area.
[0046] The organic polymer particles according to this embodiment
are usually used by dispersing in an appropriate dispersion medium.
A dispersion medium not dissolving the organic polymer particles or
not swelling the organic polymer particles is preferably used as
the dispersion medium. An aqueous medium can be given as a
preferable dispersion medium, for example. The aqueous medium here
refers to water or a mixture of water and an organic solvent
miscible with water (e.g. alcohols and alkylene glycol
derivatives).
1.2. Production of Organic Polymer Particles
1.2.1. Composition of Monomer Part
[0047] The organic polymer particles according to this embodiment
are produced by forming a polymer part obtained by polymerizing a
monomer part. Each of the monomers for forming the monomer part
will now be described.
1.2.1-1. Carboxylic Acid Monomer (A)
[0048] The types of monomer components of the carboxylic acid
monomer (A) are as described above.
[0049] The carboxylic acid monomer (A) is used in the monomer part
preferably in an amount of 2% by mass or more, and more preferably
5% by mass or more. If the amount of the carboxylic acid monomer
(A) in the monomer part is less than 2% by mass, bonding of the
primary probe may become difficult.
1.2.1-2. Other Copolymerizable Monomer (B)
[0050] Any non-crosslinkable (monofunctional) monomers and
crosslinkable (polyfunctional) monomers, or a mixture of these
monomers can be used as the other copolymerizable monomer (B).
[0051] As examples of the non-crosslinkable (monofunctional)
monomers among copolymerizable monomers (B), (meth)acrylates having
a hydrophilic functional group such as 2-hydroxyethyl acrylate,
2-hydroxyethyl methacrylate, methoxyethyl acrylate, methoxyethyl
methacrylate, polyethylene glycol monoacrylate, polyethylene glycol
monomethacrylate, glycidyl acrylate, glycidyl methacrylate,
2,3-dihydroxypropyl acrylate, and 2,3-dihydroxypropyl methacrylate;
hydrophilic monomers such as acrylamide, methacrylamide,
N-methylolacrylamide, N-methylolmethacrylamide, and
diacetoneacrylamide; aromatic vinyl monomers such as styrene,
alpha-methylstyrene, and halogenated styrene; vinyl esters such as
vinyl acetate and vinyl propionate; unsaturated nitriles such as
acrylonitrile; and ethylenically unsaturated alkyl carboxylates
such as methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl
methacrylate, butyl acrylate, butyl methacrylate, 2-ethylhexyl
acrylate, 2-ethylhexyl methacrylate, lauryl acrylate, lauryl
methacrylate, stearyl acrylate, stearyl methacrylate, cyclohexyl
acrylate, cyclohexyl methacrylate, isobornyl acrylate, and
isobornyl methacrylate can be given.
[0052] As examples of the crosslinkable (polyfunctional) monomers
among copolymerizable monomers (B), polyfunctional (meth)acrylates
such as ethylene glycol diacrylate, ethylene glycol dimethacrylate,
trimethylolpropane triacrylate, trimethylolpropane trimethacrylate,
pentaerythritol triacrylate, pentaerythritol trimethacrylate,
dipentaerythritol hexacrylate, dipentaerythritol hexamethacrylate;
conjugated diolefins such as butadiene and isoprene;
divinylbenzene, diallyl phthalate, allyl acrylate, allyl
methacrylate, and the like can be given. As further examples,
hydrophilic monomers such as polyethylene glycol diacrylate,
polyethylene glycol dimethacrylate, and poly(meth)acrylates of a
polyvinyl alcohol can be given.
[0053] Carboxylic acid monomers having a solubility in water
exceeding 20% by mass, such as acrylic acid, methacrylic acid,
maleic acid, and itaconic acid may be used as the other
copolymerizable monomer (B) in a range not hindering the effect of
the invention.
[0054] The amount of the other copolymerizable monomer (B) used is
a balance other than the above carboxylic acid monomer (A).
1.2.2. Polymerization Method
[0055] The organic polymer particles according to this embodiment
may be produced by a known method such as emulsion polymerization,
soap-free polymerization, and suspension polymerization.
Specifically, the organic polymer particles according to this
embodiment may be obtained by, for example, suspension
polymerization of the above vinyl monomer or polymer bulk
shattering. For example, the organic polymer particles according to
this embodiment can be obtained by the two-step swelling
polymerization method using seed particles (mother particles)
described in JP-UM-B-57-24369, the polymerization method described
in J. Polym. Sci., Polymer Letter Ed., 21, 937 (1983), and the
methods described in JP-A-61-215602, JP-A-61-215603, and
JP-A-61-215604. Of these, the two-step swelling polymerization
method using seed particles (nuclear particles) is preferable for
reducing the coefficient of particle size variation. Polystyrene or
a styrene-based copolymer can be used as the seed particles
(nuclear particles). The polymer part added by the two-step
swelling polymerization method consists of a homopolymer of the
above-mentioned carboxylic acid monomer (A) or a copolymer of the
carboxylic acid monomer (A) and the monomer (B).
[0056] As the emulsifier used when copolymerizing the above monomer
part, anionic surfactants such as alkyl sulfate, alkylaryl sulfate,
alkyl phosphate, and fatty acid salts; cationic surfactants such as
alkyl amine salts and alkyl quaternary amine salts; nonionic
surfactants such as polyoxyethylene alkyl ether, polyoxyethylene
alkyl aryl ether, and block polyether; amphoteric surfactants such
as carboxylic acid types (e.g. amino acids, betaine acids, and the
like) and sulphonic acid types; reactive emulsifiers with
commercial names such as Latemul S-180A.TM., and PD-104.TM.
(manufactured by KAO Corp.), Eleminol JS-2.TM. (manufactured by
Sanyo Chemical Industries, Ltd.), Aqualon HS-10.TM., KH-10.TM.,
RN-10.TM., RN-20.TM., RN-30.TM., and RN-50.TM. (manufactured by
Dai-ichi Kogyo Seiyaku Co., Ltd.), ADEKA REASOAP SE-10N.TM.,
SR-10.TM., NE-20.TM., NE-30.TM., and NE-40.TM. (manufactured by
ADEKA Corp.), and Antox MS-60.TM. (manufactured by Nippon Nyukazai
Co., Ltd.); and the like can be given. Reactive emulsifiers are
particularly preferable due to excellent particle dispersibility.
Also, polymers having a hydrophilic group with a dispersion
function may be used as the emulsifier. As examples of such
polymers, styrene-maleic acid copolymers, styrene-acrylic acid
copolymers, polyvinyl alcohols, polyalkylene glycols, sulfonated
products of polyisoprene, sulfonated products of hydrogenated
styrene-butadiene copolymers, sulfonated products of styrene-maleic
acid copolymers, sulfonated products of styrene-acrylic acid
copolymers, and the like can be given. These emulsifiers can be
used either individually or in combination of two or more. Although
there are no specific limitations to the amount of the emulsifier
to be used, the amount is usually from 0.1 to 50 parts by weight,
preferably from 0.2 to 20 parts by weight, and particularly
preferably from 0.5 to 5 parts by weight for 100 parts by weight of
the monomers used as the monomer part. An amount less than 0.1 part
by weight is not preferable due to insufficient emulsification and
decline in stability during radical polymerization. On the other
hand, an amount exceeding 50 parts by weight is not desirable due
to the problem of foaming.
[0057] As the radical polymerization initiator used in the
copolymerization of the monomer part, persulfates such as potassium
persulfate, sodium persulfate, and ammonium persulfate;
water-soluble initiators such as hydrogen peroxide, t-butyl
hydroperoxide, t-butylperoxymaleic acid, peroxide succinate, and
2,2'-azobis[2-N-benzylamidino]propane hydrochloride; oil-soluble
initiators such as benzoyl peroxide, cumene hydroperoxide,
diisopropyl peroxydicarbonate, cumyl peroxyneodecanoate, cumyl
peroxyoctoate, and azobisisobutyronitrile; redox initiators using
reducing agents such as acidic sodium sulfite, rongalite, and
ascorbic acid; and the like can be given.
1.3. Organic Polymer Particles Containing Magnetic Material and
Process for Producing the Same
[0058] The organic polymer particles according to this embodiment
may be organic polymer particles containing a magnetic material
(hereinafter referred to as "magnetic material-containing organic
polymer particles"). Since the magnetic material-containing organic
polymer particles can be separated using a magnet without using
centrifugation, for example, separating particles from samples to
be inspected can be simplified or automated.
[0059] The magnetic material-containing organic polymer particles
include (I) particles comprising a continuous phase of a
non-magnetic material such as an organic polymer with fine magnetic
material particles being dispersed therein, (II) particles
comprising a core of a secondary aggregate of fine magnetic
material particles and a shell of non-magnetic material such as an
organic polymer, (III) particles comprising mother particles, which
contain nuclear particles of a non-magnetic material such as an
organic polymer and a secondary aggregate layer (a magnetic
material layer) of fine magnetic material particles provided on the
surface of the nuclear particles, as a core, and an outermost
organic polymer layer of the mother particles, as a shell, and the
like. Of these, the above mentioned (III) particles consisting of a
core of the mother particles containing a secondary aggregate layer
of fine magnetic material particles and a shell of an organic
polymer layer are preferable. Among the organic polymers used for
the magnetic material-containing organic polymer particles with
various structures, the polymer forming the outermost surface of
the particles, excluding a core portion of the core-shell type
particles, must have a carboxyl group originating from the
carboxylic acid compound (A) having a solubility of 20% by mass or
less in water at 25.degree. C.
[0060] The most preferable magnetic material-containing organic
polymer particles have an organic polymer layer covering mother
particles containing nuclear particles and a magnetic material
layer of superparamagnetic fine particles formed on the surface of
the nuclear particles. Here, the organic polymer layer can be
obtained by the above-mentioned production process. That is, the
organic polymer layer can be obtained by polymerizing the monomer
part containing the carboxylic acid monomer (A). In addition, the
organic polymer layer is preferably a copolymer layer made from the
carboxylic acid monomer (A) and the other copolymerizable monomer
(B).
[0061] The thickness of the organic polymer layer is preferably
0.01 micrometers or more. A thickness of 0.01 micrometers or more
can prevent leakage of superparamagnetic fine particles.
[0062] As the method for producing mother particles with a magnetic
material layer of superparamagnetic fine particles on the surface
of nuclear particles, a method of dry-blending non-magnetic organic
polymer particles and superparamagnetic fine particles and
complexing these particles by physically applying a strong external
force can be given, for example. As examples of the method for
physically applying a strong force, a method of using a mortar, an
automatic mortar, or a ball mill, a blade-pressuring type powder
compressing method, a method of utilizing a mechanochemical effect
such as a mechnofusion 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 a
method for applying a strong physical adsorption force, stirring
using a vessel equipped with a stirrer having stirring blades with
a peripheral speed 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 peripheral speed of the stirring blades is slower
than 15 msec, a sufficient amount of energy for causing
superparamagnetic fine particles to be absorbed onto the surface of
the non-magnetic organic polymer 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. Fine particles of ferrite
and/or magnetite with a particle size of about 5 to 20 nm, for
example, can be preferably used as the superparamagnetic fine
particles used in the particles according to this embodiment.
[0063] A more specific method of polymerization is disclosed in
JP-A-2004-205481 and the like.
1.4. Application
[0064] The organic polymer particles according to this embodiment
can be used as an affinity carrier such as particles for chemical
compound-bonding carriers in the biochemical field, particles for
chemical-bonding carriers for diagnostics gents, and the like, and
particularly can exhibit remarkably high sensitivity as
protein-bonding particles for immunoassay bonded with a protein,
such as an antigen or an antibody, as the primary probe.
[0065] In the organic polymer particles according to this
embodiment, the substances to be inspected are biological-related
substances and chemical compounds which are contained in
immunoassay reagents and inspection samples. In the invention, the
term "biological-related substance" refers to all substances
relating to biological bodies. As examples of the
biological-related substance, substances contained in biological
bodies, substances derived from substances contained in biological
bodies, and substances which can be used in biological bodies can
be given. Examples of the biological-related substances include,
but are not limited to, proteins (e.g., enzymes, antibodies,
aptamers, and acceptors), peptides (e.g., glutathione), 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).
[0066] According to the organic polymer particles according to the
embodiment in which the polymer part having carboxyl groups is
introduced on the surface of the particles, since the carboxyl
groups are activated by known activators such as a water-soluble
carbodiimide in actual use, a primary probe can be chemically
bonded to the surface of the particles by mixing the primary probe
with the particles.
[0067] After bonding the primary probe onto the surface of the
particles, an excess amount of the primary probe is washed out and
unreacted activated carboxyl groups are deactivated as required. In
addition, after the primary probe is bonded onto the surface of the
particles, a known blocking operation may be conducted or a
blocking agent such as albumin may be used in the deactivation. A
known analytical procedure using the particles may follow.
[0068] The prove which can be supported by the organic polymer
particles according to this embodiment is a protein, preferably an
antigen or an antibody. Any antigens and antibodies reactive with a
component generally contained in samples can be used without
specific limitations. Examples which can be given include, but are
not limited to antigens or antibodies for coagulation and
fibrinolysis-related inspections such as an anti-antiplasmin
antibody for antiplasmin inspection, an anti-D-dimer antibody for
D-dimer inspection, an anti-FDP antibody for FDP inspection, an
anti-tPA antibody for tPA inspection, an anti-thrombin=antithrombin
complex antibody for TAT inspection, and an anti-FPA antibody for
FPA inspection; antigens or antibodies for tumor-related
inspections such as an anti-BFP antibody for BFP inspection, an
anti-CEA antibody for CEA inspection, an anti-AFP antibody for AFP
inspection, an anti-ferritin antibody for ferritin inspection, and
an anti-CA19-9 antibody for CA19-9 inspection; antigens and
antibodies for serum protein-related inspections such as
anti-apolipoprotein antibody for apolipoprotein inspection, an
anti-beta2-microbloblin antibody for beta2-microbloblin inspection,
an anti-alpha1-microglobulin antibody for alpha1-microglobulin
inspection, an anti-immunoglobulin antibody for immunoglobulin
inspection, an anti-CRP antibody for CRP inspection, and the like;
antigens and antibodies for endocrine function inspection such as
an anti-HCG antibody for HCG inspection; antigens and antibodies
for infection-related inspections such as an anti-HBs antibody for
HBs antigen inspection, an HBs antigen for HBs antibody inspection,
an HCV antigen for HCV antibody inspection, an HIV-1 antigen for
HIV-1 antibody inspection, an HIV-2 antigen for HIV-2 antibody
inspection, an HTLV-1 antigen for HTLV-1 inspection, a mycoplasma
antigen for mycoplasma infection inspection, a toxoplasma antigen
for toxoplasma inspection, and a streptolysin O-antigen for ASO
inspection; antigens and antibodies for autoimmune-related
inspections such as a DNA antigen for anti-DNA antibody inspection,
and a heat-denatured human IgG for RF inspection; and antigens and
antibodies for drug analysis such as an anti-digoxin antibody for
digoxin inspection and an anti-lidocaine antibody for lidocaine
inspection. As the antibody, either polyclonal antibodies or
monoclonal antibodies may be used.
2. EXAMPLES
[0069] The invention will now be described in more detail by way of
examples, which should not be construed as limiting the
invention.
2.1. Evaluation Method
2.1.1. Particle Size
[0070] The number average particle size of the particles and the
coefficient of variation were measured using a laser diffraction
particle size distribution analyzer ("SALD-200V" manufactured by
Shimadzu Corp.).
2.1.2. Carboxyl Group Content
[0071] The apparent amount of surface charge was calculated using
an aqueous dispersion containing 1 g of particles (solid component)
by conductmetric titration described in JP-A-10-270233. The amount
of background charge was calculated in the same manner using only
the dispersion medium (water). The carboxyl group content of the
particles was determined from the difference of the resulting
amounts of charges.
2.1.3. CLEIA (Chemiluminescence Enzyme Immunity Assay)
[0072] 10 microliters of particle dispersions (equivalent to 50
micrograms of particles) obtained in the later-described Examples
and Comparative Examples, sensitized with an anti-AFP antibody,
were taken in a test tube and mixed with 50 microliters of a
standard sample of an AFP antigen (manufactured by Nippon Biotest
Laboratories Inc.) diluted to a concentration of 100 ng/ml with
fetal calf serum (FCS). The mixture was reacted at 37.degree. C.
for 10 minutes. After magnetically separating the particles and
removing the supernatant liquid, 40 microliters of an anti-AFP
antibody (a reagent attached to "Lumipulse AFP-N" manufactured by
Fujirebio Inc.), labeled with an alkali phophatase (hereinafter
referred to as "ALP") as a secondary antibody, was added, followed
by a reaction at 37.degree. C. for 10 minutes. Next, after magnetic
separation and removal of the supernatant liquid, the resulting
particles were washed three times with PBS and dispersed in 50
microliters of 0.01% Tween 20. The resulting dispersion was
transferred to a new tube. After the addition of 100 microliters of
an ALP substrate solution (Lumipulse substrate solution
manufactured by Fujirebio Inc.), the mixture was reacted at
37.degree. C. for 10 minutes to measure the amount of
chemiluminescence. A chemiluminescence luminometer ("Lumat LB9507"
manufactured by Berthold Japan, Co., Ltd.) was used for measuring
the chemiluminescence.
2.2. Example 1
[0073] 2 g of a 75% di(3,5,5-trimethylhexanoyl) peroxide solution
(Peroyl 355-75(S).TM. manufactured by NOF Corp.) and 20 g of a 1%
aqueous solution of sodium dodecylsulfate were mixed and finely
emulsified using an ultrasonic dispersion machine. The emulsion was
added to a reactor containing 13 g of polystyrene particles with a
particle size of 0.77 micrometers and 41 g of water and the mixture
was stirred at 25.degree. C. for 12 hours. In another vessel, 96 g
of styrene and 4 g of divinylbenzene were emulsified in 400 g of a
0.1% aqueous solution of sodium dodecylsulfate. The resulting
emulsion was added to the above reactor. After stirring at
40.degree. C. for two hours, the mixture was heated to 80.degree.
C. and polymerized for eight hours. After cooling to room
temperature, particles were separated by centrifugation, washed
with water, dried, and ground. The ground particles were used as
nuclear particles (preparation of nuclear particles). The number
average particle size was 1.5 micrometers.
[0074] Next, ferrite-type fine magnetic material particles (average
primary particle size: 0.01 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.
[0075] Then, 15 g of the above nuclear particles and 15 g of the
hydrophobized fine magnetic material particles were thoroughly
mixed in a mixer. The mixture was processed by a hybridization
system ("Type NHS-0" manufactured by Nara Machinery Co., Ltd.) at a
peripheral blade (stirring blades) speed of 100 m/sec (16,200 rpm)
for 5 minutes to obtain mother particles with a number average
particle size of 2.0 micrometers and with a magnetic material layer
of fine magnetic material particles on the surface (preparation of
mother particles).
[0076] A 1 l separable flask was charged with 375 g of an aqueous
solution of 0.25% by mass of sodium dodecylbenzenesulfonate and
0.25% by mass of a nonionic emulsifying agent (Emulgen 150.TM.
manufactured by Kao Corp.), followed by the addition of 15 g of the
mother particles having a magnetic material layer prepared above.
The mother particles were dispersed using a homogenizer and heated
to 60.degree. C. Next, a pre-emulsion, prepared by ultrasonic
dispersion of 12 g of cyclohexylmethacrylate as a monomer part, 3 g
of 2-methacryloyloxyethyl phthalate, and 0.6 g of
di(3,5,5-trimethylhexanoyl) peroxide (Peroyl 355.TM. manufactured
by NOF Corp.) in 75 g of an aqueous solution of 0.25% by mass of
sodium dodecylbenzenesulfonate and 0.25% by mass of a nonionic
emulsifying agent (Emulgen 150.TM. manufactured by Kao Corp.), was
added dropwise to the above 500 ml separable flask controlled at
60.degree. C. over one and half hours. After heating to 75.degree.
C., the polymerization was continued for two hours before
completing the reaction. A copolymer layer covering cores of mother
particles was prepared by the above process. The particles in the
separable flask were magnetically separated and repeatedly washed
with distilled water. A dispersion of the magnetic organic polymer
particles was obtained in this manner. The resulting particles are
designated as particles (i). The solubility of
2-methacryloyloxyethyl phthalate in water at 25.degree. C. is less
than 0.2% by mass.
[0077] The particle size of the particles (i) was 2.8 micrometers
and the carboxyl group content was 9 micromol/g.
[0078] Next, an aqueous solution of
1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride
(manufactured by Dojindo Laboratories, Inc.) was added to an
aqueous dispersion of 10 mg of particles (i) with a solid
concentration of 1%. The mixture was stirred by rotation stirring
at room temperature for two hours to activate carboxyl groups.
Next, 100 micrograms of an antibody (an anti-AFP antibody,
manufactured by Cosmo Bio Co., Ltd.) to human alpha-fetoprotein
(AFP), which is a tumor marker, was added and the mixture was
reacted at room temperature for 18 hours. After the reaction, the
particles were magnetically separated, repeatedly washed with a
washing solution (25 mmol/l Tris-HCl, 7.4 pH, containing 0.01%
Tween 20), and diluted with the washing solution to a particle
concentration of 0.5% to obtain protein-bonded particles (particles
for immunoassay) with an anti-AFP antibody bonded as a primary
probe. A chemiluminescence enzyme immunity assay (CLEIA) was
carried out using the protein-bonded particles. As a result, the
signal strength of the particles (i) was found to be 152809
(RIU).
2.3. Example 2
[0079] A dispersion liquid of magnetic material-containing organic
polymer particles was obtained in the same manner as in Example 1,
except for using 2-methacryloyloxyethyl succinate instead of
2-methacryloyloxyethyl phthalate. The resulting particles are
designated as particles (ii). The solubility of
2-methacryloyloxyethyl succinate in water at 25.degree. C. is less
than 0.2% by mass.
[0080] The particle size of the particles (ii) was 2.8 micrometers
and the carboxyl group content was 12 micromol/g.
[0081] A chemiluminescence enzyme immunity assay (CLEIA) was
carried out using the particles (ii) in the same manner as the
particles (ii) in Example 1. As a result, the signal strength of
the particles (ii) was found to be 149250 (RIU).
2.4. Comparative Example 1
[0082] A dispersion liquid of magnetic material-containing organic
polymer particles was obtained in the same manner as in Example 1,
except for using methacrylic acid instead of 2-methacryloyloxyethyl
phthalate. The resulting particles are designated as particles
(i'). The solubility of methacrylic acid in water at 25.degree. C.
is 100% by mass.
[0083] The particle size of the particles (i') was 2.2 micrometers
and the carboxyl group content was 16 micromol/g.
[0084] A chemiluminescence enzyme immunity assay (CLEIA) was
carried out using the particles (i') in the same manner as the
particles (i) in Example 1. As a result, the signal strength of the
particles (i') was found to be 55591 (RIU).
[0085] It can be understood from the above results that the
particles (i) and (ii) obtained in Examples 1 and 2 have higher
sensitivity than the particles (i') obtained in Comparative Example
1 due to possession of the carboxyl group originating from the
carboxylic acid monomer (A) having a specific structure.
2.5. Reference Example 1
[0086] A dispersion liquid of magnetic material-containing organic
polymer particles was obtained in the same manner as in Example 1,
except for using 14.9 g of cyclohexylmethacrylate and 0.1 g of
2-methacryloyloxyethyl phthalate. The resulting particles are
designated as particles (ii').
[0087] The particle size of the particles (ii') was 2.8 micrometers
and the carboxyl group content was 2 micromol/g.
[0088] A chemiluminescence enzyme immunity assay (CLEIA) was
carried out using the particles (ii') in the same manner as the
particles (ii) in Example 1. As a result, the signal strength of
the particles (ii') was found to be 56443 (RIU).
[0089] Although only some embodiments of the invention have been
described in detail above, those skilled in the art would readily
appreciate that many modifications are possible in the embodiments
without materially departing from the novel teachings and
advantages of the invention. Accordingly, such modifications are
intended to be included within the scope of the invention.
* * * * *