U.S. patent application number 13/030089 was filed with the patent office on 2011-06-09 for method for detecting target substance and target-substance detection kit.
This patent application is currently assigned to CANON KABUSHIKI KAISHA. Invention is credited to Takashi Ikeda, Takeshi Imamura, Masato Minami, Kazumichi Nakahama, Miki Ogawa.
Application Number | 20110136264 13/030089 |
Document ID | / |
Family ID | 38563770 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110136264 |
Kind Code |
A1 |
Ogawa; Miki ; et
al. |
June 9, 2011 |
METHOD FOR DETECTING TARGET SUBSTANCE AND TARGET-SUBSTANCE
DETECTION KIT
Abstract
The present invention provides a method for detecting a target
substance by detecting the presence or concentration of a target
substance in a sample solution by bringing the sample solution into
contact with a detecting element including a detecting part and a
non-detecting part and detecting the presence or number of a
magnetic label (magnetic marker) present in the vicinity of the
surface of the detecting part and provides a target-substance
detection kit. The surface potential .psi..sub.1 of the magnetic
label in the sample solution, the surface potential .psi..sub.2 of
the detecting part, and the surface potential .psi..sub.3 of the
non-detecting part satisfy any one of the following relationships
i) to iv): .psi..sub.1.psi..sub.3>0 and .psi..sub.2=0, i)
.psi..sub.1.psi..sub.2<0 and .psi..sub.3=0, ii)
.psi..sub.1.psi..sub.2<0, .psi..sub.2.psi..sub.3>0, and
|.psi..sub.2|>|.psi..sub.3|, and iii)
.psi..sub.1.psi..sub.2<0 and .psi..sub.2.psi..sub.3<0; iv)
and the target substance borne by the magnetic label is captured by
a primary capturing body borne by the detecting part, or the target
substance captured by a primary capturing body borne by the
detecting part is captured by a secondary capturing body borne by
the magnetic label.
Inventors: |
Ogawa; Miki; (Machida-shi,
JP) ; Minami; Masato; (Yokohama-shi, JP) ;
Imamura; Takeshi; (Chigasaki-shi, JP) ; Ikeda;
Takashi; (Yokohama-shi, JP) ; Nakahama;
Kazumichi; (Tokyo, JP) |
Assignee: |
CANON KABUSHIKI KAISHA
Tokyo
JP
|
Family ID: |
38563770 |
Appl. No.: |
13/030089 |
Filed: |
February 17, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12162789 |
Jul 30, 2008 |
7932100 |
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|
PCT/JP2007/057712 |
Mar 30, 2007 |
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13030089 |
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Current U.S.
Class: |
436/518 |
Current CPC
Class: |
Y10S 436/807 20130101;
G01N 33/54373 20130101 |
Class at
Publication: |
436/518 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2006 |
JP |
2006-100683 |
Nov 24, 2006 |
JP |
2006-317401 |
Claims
1.-10. (canceled)
11. A method for detecting a target substance in a sample solution,
comprising: a first step of preparing a detecting element
comprising a detecting part and a non-detecting part, and a
magnetic label including the target substance, the detecting part
including a primary capturing body on the surface thereof; a second
step of capturing the target substance of the magnetic label with
the primary capturing body; and a third step of detecting the
magnetic label present in the vicinity of the detecting part,
wherein the surface potential .psi..sub.1 of the magnetic label in
the second step, the surface potential .psi..sub.2 of the detecting
part, and the surface potential .psi..sub.3 of the non-detecting
part satisfy any one of the following relationships i) to iv) by
forming a layer on a surface of at least one of the magnetic label,
the detecting part, and the non-detecting part in the first step:
.psi..sub.1.psi..sub.3>0 and .psi..sub.2=0, i)
.psi..sub.1.psi..sub.2<0 and .psi..sub.3=0, ii)
.psi..sub.1.psi..sub.2<0, .psi..sub.2.psi..sub.3>0, and
|.psi..sub.2|>|.psi..sub.3|, and iii)
.psi..sub.1.psi..sub.2<0 and .psi..sub.2.psi..sub.3<0.
iv)
12. The method for detecting a target substance according to claim
11, wherein the surface potential of the detecting part is
generated by applying a voltage or current by an external power
supply connected to the detecting part.
13. The method for detecting a target substance according to claim
11, wherein the magnetic particle comprises a secondary capturing
body that captures the target substance.
14. A method for detecting a target substance in a sample solution,
comprising: a first step of preparing a detecting element
comprising a detecting part and a non-detecting part, and a
magnetic label including a secondary capturing body, the detecting
part including a primary capturing body on the surface thereof; a
second step of capturing the target substance with the secondary
capturing body of the magnetic label after capturing the target
substance with the primary capturing body; and a third step of
detecting the magnetic label present in the vicinity of the
detecting part, wherein the surface potential .psi..sub.1 of the
magnetic label in the second step, the surface potential
.psi..sub.2 of the detecting part, and the surface potential
.psi..sub.3 of the non-detecting part satisfy any one of the
following relationships i) to iv) by forming a layer on a surface
of at least one of the magnetic label, the detecting part, and the
non-detecting part in the first step: .psi..sub.1.psi..sub.3>0
and .psi..sub.2=0, i) .psi..sub.1.psi..sub.2<0 and
.psi..sub.3=0, ii) .psi..sub.1.psi..sub.2<0,
.psi..sub.2.psi..sub.3>0, and |.psi..sub.2|>|.psi..sub.3|,
and iii) .psi..sub.1.psi..sub.2<0 and
.psi..sub.2.psi..sub.3<0. iv)
15. The method for detecting a target substance according to claim
14, wherein the surface potential of the detecting part is
generated by applying a voltage or current by an external power
supply connected to the detecting part.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for detecting a
target substance in a sample solution and relates to a
target-substance detection kit. Specifically, the present invention
relates to a method for detecting a target substance and a
target-substance detection kit which can be suitably applied to a
so-called biosensor utilizing the specific molecular recognition
ability of a substance of biological origin or its analogue.
BACKGROUND ART
[0002] Biosensors are a measurement device utilizing excellent
molecular recognition ability of living organisms or biomolecules.
As pairs of affinity binding partners in living organisms, for
example, enzyme-substrate, antigen-antibody, and DNA-DNA are known.
Biosensors utilize the principle that one of these pairs can be
selectively measured by using the other of the pair immobilized or
supported on a substrate. Recently, biosensors have been expected
to be broadly used not only in the field of medicine but also in
the fields of environment and food. Consequently, in order to
broaden the application fields of biosensors, highly sensitive and
highly efficient biosensors which can be installed at every place
or which are reduced in size and weight so as to be portable are
expected.
[0003] As one of methods detecting such biomolecular interactions,
the magnetic detection method utilizing a magnetic label is now
under active development and used in solid-phase analysis.
[0004] FIG. 1 illustrates an example of the conventional
solid-phase analysis using a magnetic label. In the method shown in
FIG. 1, a primary capturing body 3 (called a primary antibody when
an antigen is detected by utilizing an antigen-antibody reaction)
which can specifically recognize and capture a region (called an
epitope when the target substance is an antigen and the antigen is
detected by utilizing an antigen-antibody reaction) of a target
substance 5 is previously immobilized on a surface of a substrate
1. Then, a sample solution containing the target substance 5 is
brought into contact with the surface of the substrate 1. With this
process, the target substance 5 is specifically captured by the
primary capturing body 3. Then, a magnetic label 9 provided with a
secondary capturing body 4 (called a secondary antibody when an
antigen is detected by utilizing an antigen-antibody reaction) is
added to the sample solution. The secondary capturing body 4 can
specifically recognize and capture a region, of the target
substance 5, other than the region which is specifically captured
by the primary capturing body 3. (Here, the magnetic label 9
includes a magnetic structure 2 and a secondary capturing body 4
immobilized on the surface of the magnetic structure 2.) With this
process, the secondary capturing body 4 recognizes and captures the
target substance 5 specifically captured by the primary capturing
body 3 immobilized on the surface of the substrate 1. Consequently,
the magnetic label is apparently captured by the target substance.
Accordingly, as shown in FIG. 1, the magnetic label is immobilized
in the vicinity of the surface of the substrate 1 via the target
substance 5.
[0005] In addition, as a method different from the above, the
following method is also known. A magnetic label 2 provided with a
secondary capturing body 4 is added to a sample solution containing
the target substance 5 to form a complex of "the target substance
and the secondary capturing body on the magnetic label". Then, the
resulting complex is brought into contact with a primary capturing
body 3 immobilized on a substrate 1. As a result, as shown in FIG.
1, the magnetic label can be immobilized on the surface of the
substrate via the target substance.
[0006] Lastly, the number of the magnetic label immobilized on the
surface of a detecting element is measured by any method and
thereby the number or concentration of the target substance to be
determined can be calculated.
[0007] As a target-substance detecting element using such a
magnetic detection technology, Japanese Patent Application
Laid-Open No. 2001-033455 discloses an immunoassay for detecting a
target substance by using a magnetic material as a label. The label
is bound to the target substance in a sample solution by an
antigen-antibody reaction and is magnetized and detected using a
superconducting quantum interference device (SQUID) as a magnetic
sensor.
[0008] Further, International Publication No. WO 03/067258
discloses a biosensor for analyzing an object to be measured using
detecting elements for detecting a magnetic field produced by bound
magnetic molecules and having semiconductor hall devices. The
analysis is conducted based on the amount of the specified magnetic
molecules.
[0009] U.S. Pat. No. 5,981,297 discloses a method for detecting a
magnetic signal of fine magnetic particles using a magnetoresistive
element. A primary capture molecule on a sensor element is bound to
a secondary capture molecule labeled with the fine magnetic
particles as a signal via a target molecule.
[0010] The above-described methods are biosensing methods utilizing
magnetic labels. In the meantime, Japanese Patent Application
Laid-Open No. 2005-91014 discloses a biosensing method using a
substrate provided with a biomolecule-immobilizing region and a
template region surrounding the biomolecule-immobilizing region.
The template region is covered with a monomolecular layer which
does not react with the target molecule and a capture molecule.
Such a structure is aimed to stably generate a signal which is
derived from biomolecular interaction between a target molecule and
a capture molecule and to detect the signal with a high accuracy
and high sensitivity.
[0011] However, biosensors still have challenges to achieve further
higher sensitivity and accuracy (a decrease in noise) and to reduce
reaction time, namely, to improve the detection efficiency.
DISCLOSURE OF THE INVENTION
[0012] The present invention provides a method for detecting a
target substance with excellent detection efficiency and a high
sensitivity and provides a target-substance detection kit.
[0013] The present invention provides a method for detecting a
target substance in a sample solution by using a detecting element
including a detecting part provided with a primary capturing body
on the surface thereof and a non-detecting part and by detecting a
magnetic label present in the vicinity of the detecting part.
[0014] In the method according to the present invention, the
surface potential .psi..sub.1 of the magnetic label in the sample
solution, the surface potential .psi..sub.2 of the detecting part,
and the surface potential .psi..sub.3 of the non-detecting part
satisfy any one of the following relationships i) to iv):
.psi..sub.1.psi..sub.3>0 and .psi..sub.2=0, i)
.psi..sub.1.psi..sub.2<0 and .psi..sub.3=0 ii)
.psi..sub.1.psi..sub.2<0, .psi..sub.2.psi..sub.3>0 and
|.psi..sub.2|>|.psi..sub.3|, and iii)
.psi..sub.1.psi..sub.2<0 and .psi..sub.2.psi..sub.3<0; and
iv)
[0015] the target substance borne by the magnetic label is captured
by the primary capturing body borne by the detecting part, or the
target substance captured by the primary capturing body borne by
the detecting part is captured by a secondary capturing body borne
by the magnetic label.
[0016] The present invention further provides a target-substance
detection kit for detecting a target substance by detecting a
magnetic label present in the vicinity of a detecting part of a
detecting element including the detecting part provided with a
primary capturing body on the surface thereof and a non-detecting
part.
[0017] In the kit according to the present invention, the surface
potential .psi..sub.1 of the magnetic label in the sample solution,
the surface potential .psi..sub.2 of the detecting part, and the
surface potential .psi..sub.3 of the non-detecting part satisfy any
one of the following relationships i) to iv):
.psi..sub.3>0 and .psi..sub.2=0, i)
.psi..sub.1.psi..sub.2<0 and .psi..sub.3=0, ii)
.psi..sub.1.psi..sub.2<0, .psi..sub.2.psi..sub.3>0 and
|.psi..sub.2|>|.psi..sub.3|, and iii)
.psi..sub.1.psi..sub.2<0 and .psi..sub.2.psi..sub.3<0; and
iv)
[0018] the target substance borne by the magnetic label is captured
by the primary capturing body borne by the detecting part, or the
target substance captured by the primary capturing body borne by
the detecting part is captured by a secondary capturing body borne
by the magnetic label.
[0019] The relationships i) to iv) are preferably satisfied by
forming a layer on any surface of the magnetic label, the detecting
part, and the non-detecting part the surface potentials of which do
not satisfy any one of relationships i) to iv).
[0020] Preferably, the magnetic label and the non-detecting part
each have such a layered structure and the outermost layer of the
magnetic label having the layered structure and the outermost layer
of the non-detecting part having the layered structure are formed
of the same material.
[0021] The layers formed of the same material are preferably formed
of a graft polymer.
[0022] The surface potential of the detecting part is preferably
generated by a voltage or current supplied from a power supply
source connected to the detecting part.
[0023] The detecting part preferably has a terminal for applying a
voltage or current.
[0024] Further futures of the present invention will become
apparent from the following description of exemplary embodiments
with reference to the attached drawings.
BRIEF DESCRIPTION OF THE DRAWING
[0025] FIG. 1 is a diagram illustrating the conventional
solid-phase analysis using a magnetic label;
[0026] FIG. 2 is a schematic diagram illustrating a relationship
between a magnetic label and a detecting element according to an
embodiment;
[0027] FIG. 3 is a schematic diagram illustrating a relationship
between a detecting element and a magnetic label according to an
embodiment, the detecting element including a non-detecting part
and a detecting part and the magnetic label being provided with a
target substance on the surface thereof;
[0028] FIG. 4 is a schematic diagram illustrating a relationship
between a detecting element and a magnetic label according to an
embodiment, the detecting element including a non-detecting part
and a detecting part and the magnetic label being provided with a
secondary capturing body capturing a target substance;
[0029] FIG. 5 is a schematic diagram illustrating a relationship
between a detecting element and a magnetic label according to an
embodiment, the detecting element including a detecting part and a
non-detecting part having a coating layer on the surface
thereof;
[0030] FIG. 6 is a schematic diagram illustrating a relationship
between a detecting element and a magnetic label according to an
embodiment, the detecting element including a detecting part and a
non-detecting part having a coating layer on the surface thereof
and the magnetic layer having a coating layer on the surface
thereof;
[0031] FIG. 7 is a schematic diagram illustrating a detecting
element according to an embodiment in which the polarity of the
surface potential of a detecting part can be controlled by applying
a voltage or current;
[0032] FIG. 8 is a schematic diagram illustrating a detecting
element and magnetic label in Example 1;
[0033] FIG. 9 is a schematic diagram illustrating a detecting
element and magnetic label in Examples 2 to 5;
[0034] FIG. 10 is a schematic diagram illustrating a magnetic label
according to an embodiment; and
[0035] FIG. 11 is a schematic diagram illustrating a
magnetoresistive element according to an embodiment.
BEST MODE FOR CARRYING OUT THE INVENTION
[0036] FIGS. 2 to 4 are drawings schematically illustrating
magnetic labels and detecting elements used for detection methods
according to embodiments of the present invention, the magnetic
labels and detecting elements being classified according to the
difference in the target-substance detection methods.
[0037] Reference numeral 2 denotes a magnetic structure, reference
numeral 3 denotes a primary capturing body, reference numeral 4
denotes a secondary capturing body, reference numeral 5 denotes a
target substance, reference numeral 7 denotes a detecting part,
reference numeral 8 denotes a non-detecting part, and reference
numeral 9 denotes a magnetic label. In these embodiments, the
magnetic label 9 includes a magnetic structure 2 and a secondary
capturing body 4 provided on the surface thereof. FIG. 2
illustrates a configuration in which a magnetic label is provided
with a secondary capturing body on the surface thereof, FIG. 3
illustrates a configuration in which a magnetic label is provided
with a target substance on the surface thereof, and FIG. 4
illustrates a configuration in which a magnetic label is provided
with a secondary capturing body capturing a target substance on the
surface thereof.
[0038] The configuration shown in FIG. 2 and the configuration
shown in FIG. 4 are the same in that a magnetic label is provided
with a secondary capturing body on the surface thereof. However,
from the viewpoint of a target-substance detection method, the
configuration shown in FIG. 2 is one that a target substance
captured by a primary capturing body is captured by a secondary
capturing body borne by the magnetic label, but the configuration
shown in FIG. 4 is one that a target substance borne by a magnetic
label is captured by a primary capturing body. Similarly, from the
viewpoint of a target-substance detection method, the configuration
shown in FIG. 3 and the configuration shown in FIG. 4 are the same
in that a target substance borne by a magnetic label is captured by
a primary capturing body. In addition, the structure of the
detecting part and the non-detecting part may be one shown in FIG.
5. In the structure shown in FIG. 5, a substrate 6 contains a
region serving as a part of a detecting part and a region serving
as a part of a non-detecting part. In a structure such as that
shown in FIG. 5, the detecting part 7 includes a region of a
substrate 1 for immobilizing a primary capturing body 3 on the
surface thereof and the primary capturing body 3, and the
non-detecting part 8 includes a region other than the detecting
part 7 of the substrate 1 and a layer formed on the surface of the
region. In addition, in the present invention, the substrate
functions as a support for the entire detecting element. The
substrate refers to a detecting element which is not yet provided
with a primary capturing body. Therefore, the substrate is a part
of the substrate, and when the substrate is constituted with one
layer, the substrate is identical with the substrate.
[0039] Furthermore, in the embodiment shown in FIG. 5, a plurality
of layers are formed on the surface of the substrate 6 at a region
other than the region serving as the detecting part 7. In the
drawings, the outermost layer among the plurality of layers is
shown as a coating layer 16. The magnetic label may have a
structure, as shown in FIG. 6, in which a coating layer 16 is
provided on the surface of a magnetic structure 2 and a secondary
capturing body 4 is provided on the coating layer.
[0040] In the present invention, a sample solution is brought into
contact with a detecting element including a detecting part and a
non-detecting part. The presence or concentration of a target
substance in the sample solution is measured by detecting the
presence or the number of the magnetic label (magnetic marker)
remaining in the vicinity of the surface of the detecting part.
[0041] On this occasion, it is an important characteristic of the
present invention that the surface potentials of the detecting
part, the non-detecting part, and the magnetic label when they are
in contact with the sample solution are in a relationship shown
below. Further, in the present invention, the term "vicinity"
denotes a range of 1 mm or less.
[0042] That is, the surface potential .psi..sub.1 of the magnetic
label in the sample solution, the surface potential .psi..sub.2 of
the detecting part, and the surface potential .psi..sub.3 of the
non-detecting part satisfy any one of the following relationships
i) to iv):
.psi..sub.1.psi..sub.3>0 and .psi..sub.2=0, i)
.psi..sub.1.psi..sub.2<0 and .psi..sub.3=0, ii)
.psi..sub.1.psi..sub.2<0, .psi..sub.2.psi..sub.3>0 and
|.psi..sub.2|>|.psi..sub.3|, and iii)
.psi..sub.1.psi..sub.2<0 and .psi..sub.2.psi..sub.3<0; and
iv)
[0043] the target substance borne by the magnetic label is captured
by a primary capturing body borne by the detecting part, or the
target substance captured by a primary capturing body borne by the
detecting part is captured by a secondary capturing body borne by
the magnetic label.
[0044] With such a characteristic, the magnetic label can be
rapidly and selectively led to the vicinity of the detecting part
and the detection efficiency can be improved.
[0045] In the present invention, the term "surface potential"
refers to a charging condition of a surface. The "surface potential
of the A" denotes the condition of the electrified surface of the A
as a whole, not a local condition of the electrified surface of the
A.
[0046] Therefore, the surface potential of the A may be the average
surface charge of the A or may be the average surface potential of
the A. Further, the surface potential may be the zeta potential of
the A. Furthermore, the surface potential may be the potential
generated on the surface by applying a potential to the surface of
the A by an external power supply. The external power supply may be
connected to a reaction vessel as long as the surface potential of
the detecting part can be controlled. Further, the reaction vessel
itself may have a surface potential whose polarity different from
that of the surface potential of the detecting part.
[0047] When an average surface charge is used as the surface
potential, the surface of the A is provided with a compound
containing a functional group which can become an anion or a
functional group which can become a cation in a solution. In such a
case, the surface potential refers to the average charge of the
compound as a whole.
[0048] Therefore, the repulsive force between two materials having
surface potentials is larger when the polarities of the electric
potentials are the same and the absolute values is large.
[0049] Hereinafter, the magnetic label, the detecting element, and
the detection kit according to the present invention will be
described. Then, the conditions in the respective relationships i)
to iv) will be described in detail.
[0050] In the present invention, the magnetic label is rapidly and
selectively led to the vicinity of the detecting part by
controlling the surface potentials of the detecting part, the
non-detecting part, and the magnetic label.
<Target-Substance Detecting Element>
[0051] The target-substance detecting element used in the present
invention includes a detecting part provided with a primary
capturing body on the surface thereof and a non-detecting part. A
target substance is captured by the primary capturing body and,
consequently, a magnetic label is immobilized in the vicinity of
the detecting part and the detecting part recognizes the magnetic
label. Thus, the detection of the target substance is carried out
by utilizing a change in the signal due to the presence of the
magnetic label.
[0052] In the present invention, the detecting part of the
detecting element refers to a part having a function measuring the
presence or amount of the target substance in a sample solution
based on the presence or amount of the magnetic label. The
detecting part provided with a primary capturing body for capturing
the target substance on the surface thereof. The non-detecting part
refers to a part other than the detecting part in the detecting
element. As long as the non-detecting part and the detecting part
of the detecting element are adjacent to each other, they may be
partially in contact with each other or the non-detection may be
disposed so as to surround the detecting part.
[0053] If the surface potentials of the detecting part and the
non-potential part with respect to the surface potential of the
magnetic label satisfy any one of the relationships i) to iv) when
a primary capturing body of the detecting part captures the target
substance borne by the magnetic label or a secondary capturing body
of the magnetic label captures the target substance captured by a
primary capturing body of the detecting part, the detecting part
and the non-detecting part may be constituted with one layer or may
be a layered structure of a plurality of layers. The surface
potential of the detecting part or the non-detecting part may be
controlled by the property of the material forming the surface of
the detecting part or the non-detecting part or may be controlled
by applying a voltage or current to one of the detecting part and
the non-detecting part or both by connecting to an external power
supply. In such a case, the detecting part or the non-detecting
part has a terminal for applying a voltage or current by an
external power supply.
[0054] When the surface potential is controlled by the material
forming the surface of the detecting part or the non-detecting part
and the detecting part or the non-detecting part is formed into one
layer, a material whose surface potential satisfies the
above-mentioned conditions is preferably used as the substrate.
Further, the detecting part and the non-detecting parts are formed
into one layer, the detecting part and the non-detecting part are
formed of different materials. On the other hand, when the surface
potential is controlled by the material forming the surface of the
detecting part or the non-detecting part and the detecting part or
the non-detecting part is a layered structure of a plurality of
layers, a material whose surface potential satisfies the
above-mentioned conditions is preferably used as the outermost
layer. Examples of the material which satisfies the above-mentioned
conditions include a polymer containing a functional group which
becomes an anion or a functional group which becomes a cation and
an inorganic oxide having an isoelectric point in a solution at a
specific temperature or pH. An example of the functional group
which becomes an anion in a solution at a specific temperature or
pH is a carboxyl group, and an example which becomes a cation is an
amino group. In addition, as long as the surface potential of the
outermost layer of a layered structure satisfies the
above-mentioned conditions, layers other than the outermost layer
may be formed of a material not having a surface potential or a
material having a surface potential which does not satisfy the
above-mentioned conditions. Further, when the material forming the
outermost layer does not satisfy the above-mentioned conditions as
a single material, the outermost layer may be formed of such a
material as long as the layered structure satisfies the
above-mentioned conditions.
[0055] Further, when the surface potential is controlled by
applying a voltage or current by connecting the detecting part or
the non-detecting part to an external power supply, a material not
having a surface potential or having a surface potential which does
not satisfy the above-mentioned conditions when the external power
supply is not connected may be used as long as the surface
potential can be controlled to the above-mentioned conditions by
applying the surface potential to the detecting part or the
non-detecting part by the external power supply. FIG. 7 illustrates
an embodiment in which the detecting part is connected to an
external power supply. In this embodiment, a potential is added to
the surface of the detecting part by the external power supply 15.
In this method, preferably, the surface potential of the detecting
part 7 can be readily altered according to the surface potential of
the magnetic label.
[0056] Further, preferably, a molecule having an active group is
previously immobilized on a part of the substrate surface serving
as a part of the detecting part, and a primary capturing body
serving as another part of the detecting part is immobilized via
the active group.
(Capturing Body)
[0057] The capturing body used in the present invention is a
material which is involved in the selection of a target substance
in a sample solution and can be selected according to the target
substance.
[0058] Here, the target substance in the present invention will be
described in advance of the description of the capturing body in
the present invention.
[0059] The target substance is contained in a sample solution which
is reacted with a detecting element. The target substance is
captured at least by a primary capturing body on the surface of a
detecting part. The target substance in the present invention is
typically a chemical material (biological material) present in the
body of an organism. In the present invention, generally, the
object to be detected itself is the target substance.
[0060] Actually, in the present invention, an object to be detected
may be detected by using a target substance. Therefore, the object
to be detected itself is the target substance as described above
and may be directly detected by capturing the object by a capturing
body, or the object to be detected is different from the target
substance and may be indirectly detected by capturing the target
substance by a capturing body. An example of the latter is a case
that a target substance is generated by the presence of an object
to be detected. Therefore, the object to be detected is not limited
to biological materials and the size of the object is not limited.
However, preferable examples of the target substance are biological
materials such as sugars, proteins, amino acids, antibodies,
antigens, pseudo-antigens, vitamins, genes, related materials
thereof, and artificially synthesized biomimetic materials. In
addition, the sample solution may be a specimen itself containing
an object to be determined or may be prepared by treating a
specimen by various processes such as extraction, separation,
dilution, or purification. The sample solution is prepared by using
a liquid solvent, such as water, buffer, or mixture of water and a
water-soluble organic solvent, depending on the type of a target
substance.
[0061] The capturing bodies used in the present invention are
materials which can capture the above-mentioned target substances
referred to as examples, examples of which include proteins such as
enzymes, antibodies, and antigens; DNAs; RNAs; and sugar chains,
but not limited thereto.
[0062] Examples of a combination of a target substance and a
primary or secondary capturing body in the present invention
include antigen-antibody, enzyme-substrate, DNA-DNA, DNA-RNA,
DNA-protein, RNA-protein, and sugar chain-protein, but not
specifically limited thereto as long as the combination is in a
specific binding. The above shows the combination. Therefore, when
a combination of a target substance and a capturing body is
expressed as "A-B", it means both cases that the A denotes a target
substance and the B denotes a capturing body and that the B denotes
a target substance and the A denotes a capturing body.
<Target-Substance Detection Kit>
[0063] The target-substance detection kit according to this
embodiment includes a magnetic label and a target-substance
detecting element. The target-substance detecting element consists
of the above-described target-substance detecting element.
(Magnetic Label)
[0064] A magnetic label used in the present invention includes at
least a magnetic structure and a secondary capturing body for
capturing a target substance on the surface of the magnetic
structure, or a complex of a secondary target substance and target
substance. That is, there are cases that the magnetic label
includes a magnetic structure and a secondary capturing body on the
surface of the magnetic structure and that the magnetic label
includes a magnetic structure, a secondary capturing body on the
surface of the magnetic structure, and a target substance captured
by the secondary capturing body. The magnetic label is required to
have a surface potential when the target substance borne by the
magnetic label is captured by a primary capturing body of a
detecting part or when the secondary capturing body borne by the
magnetic label captures a target substance captured by a primary
capturing body borne by a detecting part. Furthermore, the magnetic
label is required to satisfy physical properties or characteristics
as a label for detecting a target substance. Therefore, the
magnetic structure may be selected from generally used fine
magnetic particles (magnetic beads) exhibiting paramagnetism or
superparamagnetism.
[0065] Examples of a magnetic material constituting the magnetic
structure include metal oxides. Metal oxides are readily charged in
an aqueous solution, namely, are positively charged at a pH lower
than the isoelectric point and are negatively charged at a pH
higher than the isoelectric point and, therefore, are preferable.
Among metal oxides, particularly, particles of iron oxides such as
ferrite and magnetite which are generally used as magnetic
structures of magnetic labels exhibit sufficient magnetism under
bioactive conditions and are negligibly degraded, such as
oxidation, in a solvent. Therefore, iron oxide particles are
preferable. Ferrite is selected from magnetite (Fe.sub.3O.sub.4),
maghemite (.gamma.-Fe.sub.2O.sub.3), and complexes thereof obtained
by substituting a part of Fe with another atom. Examples of the
another atom include Li, Mg, Al, Si, Ca, Sc, Ti, V, Cr, Mn, Co, Ni,
Cu, Zn, Ga, Ge, Zr, Nb, Mo, Cd, In, Sn, Ta, and W.
[0066] The magnetic structure may be core-shell particles which are
formed by using a substrate consisting of a magnetic material as a
core and forming a polymer layer (resin layer) on the surface of
the substrate for improving dispersibility. The resin layer may be
formed, for example, of a styrene-based, dextran-based, or
acrylamide-based resin. Here, the styrene-based resin is defined as
a resin consisting of styrene and styrene derivatives. The
dextran-based resin and the acrylamide-based resin are similarly
defined. In addition, a resin obtained by copolymerizing at least
two monomers forming a styrene-based resin, a dextran-based resin,
or an acrylamide-based resin is also contained in a styrene-based,
dextran-based, or acrylamide-based resin.
[0067] When a resin layer formed as described above has a surface
potential in a sample solution and thereby the magnetic label has a
surface potential, a layered structure composed of a metal oxide
and a resin layer can be used in a magnetic label as the magnetic
structure.
[0068] Further, when the resin layer does not have a sufficient
surface potential in a sample solution, a layered structure
prepared by using the core-shell particle as a substrate and
further forming a coating layer described below on the surface of
the substrate may be used. In addition, besides the core-shell type
particles, particles prepared by dispersing fine particles composed
of a magnetic material in a styrene-based, dextran-based, or
acrylamide-based resin or particles supporting fine particles
composed of a magnetic material on the surfaces of resin particles
may be used as the magnetic structure of the present invention. As
these magnetic structures, for example, Dynabeads is commercially
available from Dynal, Micromer-M and Nanomag-D are commercially
available from Micromod, and Estapor is commercially available from
Merk.
[0069] The size of the magnetic structure may variously vary
depending on the shape, size, or use of a detecting element. In
general, the diameter of a magnetic structure is preferably 3 nm to
500 .mu.m, more preferably 3 nm to 10 .mu.m, and further preferably
5 nm to 1 .mu.m. The diameter or average particle size of the
magnetic structure can be measured by a dynamic light scattering
method.
[0070] The magnetic label is preferably provided with a secondary
capturing body for capturing a target substance on the surface of
the magnetic structure. Further, preferably, a primary capturing
body of a detecting part specifically recognizes a primary region
of a target substance in a sample solution and captures the target
substance through the primary region, and the secondary capturing
body of the magnetic label specifically recognizes a secondary
region of the target substance in the sample solution and captures
the target substance through the secondary region. Here, the
secondary region is different from the primary region and is at
least an area of a region other than the primary region of the
target substance. In this case, both the magnetic label and the
detecting part each have a capturing body on the surface thereof,
and the target substance is captured by both the primary capturing
body on the surface of the magnetic label and the secondary
capturing body on the surface of the detecting part. Consequently,
the magnetic label is immobilized in the vicinity of the surface of
the detecting part via the target substance. The detecting part
detects the immobilized magnetic label and the target substance can
be readily detected.
[0071] Further, when a magnetic label is provided with a target
substance on the surface thereof, the target substance can be
detected by capturing the target substance by a capturing body on
the surface of a detecting part even if the magnetic label is not
provided with a secondary capturing body on the surface. In such a
case, the target substance on the surface of the magnetic label
preferably includes at least two regions, i.e., a region where the
target substance is immobilized to the magnetic label and a region
where the target substance is captured by the primary capturing
body on the surface of the detecting part. Further, when a magnetic
label includes a complex of a target substance and a secondary
capturing body, the target substance can be detected by capturing
the target substance by a primary capturing body borne by a
detecting part. In this specification and the present invention,
the concept that a magnetic label is provided with a target
substance on the surface of a magnetic structure includes a case
that a magnetic label is provided with a complex of a target
substance and a secondary capturing body as a result that the
secondary capturing body has captured the target substance.
[0072] The above-described detecting element and the magnetic label
can constitute a target-substance detection kit. That is, the kit
can be used for detecting the presence or concentration of a target
substance in a sample solution by bringing the sample solution into
contact with the surface of a detecting part of the detecting
element to lead the target substance to the detecting part and
detecting the presence or number of the magnetic label present in
the vicinity of the surface of the detecting part.
(Coating Layer)
[0073] The magnetic label and the detecting element according to
the present invention may each have a layered structure composed of
a plurality of layers, as described above. The layered structure
composed of a plurality of layers can be prepared, for example, by
forming at least one layer on the surface of a substrate. In such a
case, the outermost layer of the layered structure composed of a
plurality of layers containing the substrate is called a coating
layer. Therefore, when one layer is formed on the surface of a
substrate, the layer is a coating layer. In the present invention,
a substrate or a magnetic structure in a state before the formation
of a coating layer or a resin layer is called a substrate for the
convenience of description.
[0074] The surface potential of the substrate surface can be
controlled by forming a coating layer. Therefore, the relationships
i) to iv) can be satisfied by forming a coating layer on any one of
the surfaces of the magnetic label, the detecting part, and the
non-detecting part even if the magnetic label, the detecting part,
and the non-detecting part do not satisfy any of the relationships
i) to iv). Even if the substrates of the magnetic label and the
detecting element are composed of a material which exhibits a zero
or near-zero surface potential in a sample solution, the effect of
the present invention can be obtained. Any materials can be used as
a coating layer as long as the material can exhibit a surface
potential in a sample solution according to purpose. A material
selected from, for example, inorganic materials such as glass,
organic materials such as resins, semiconducting materials such as
silicon, and metal materials can be used according to purpose.
[0075] Further, when the target substance or the capturing body is
a biological material, these coating layers are preferably a
hydrophilic layer.
[0076] Generally, biological materials such as proteins are
hydrophobic. Therefore, when the coating layer is hydrophobic,
non-specific adsorption to the coating layer is readily caused by
"hydrophobic interaction".
[0077] For example, if a target substance is non-specifically
adsorbed to the surface of a detecting part at a region other than
a primary capturing body, a secondary capturing body on the surface
of a magnetic label captures the non-specifically adsorbed target
substance and thereby the signal of the magnetic label occurs as a
noise. This noise may cause a decrease in the detection sensitivity
or accuracy. In addition, when a secondary capturing body borne by
the surface of a magnetic label or a target substance borne by the
surface of a magnetic label is non-specifically adsorbed directly
to a non-detecting part or a detecting part without the presence of
a target substance, a decrease in the detection sensitivity or
accuracy may be similarly caused.
[0078] On the other hand, when the coating layer is hydrophilic,
"hydrophobic interaction", which is one cause of non-specific
adsorption of biological materials, can be reduced. Thus,
non-specific adsorption of biological materials can be decreased.
The hydrophilic coating layer is formed of, for example, a graft
polymer such as polyglycidyl methacrylate, PHEMA
(poly(2-hydroxyethyl methacrylate)), or polyethylene glycol
methacrylate. Among them, polyglycidyl methacrylate is excellent in
the prevention of non-specific adsorption of biological materials
such as proteins. Further, a functional group such as an amino
group can be introduced into polyglycidyl methacrylate by opening
the epoxy group. Therefore, polyglycidyl methacrylate is preferable
as a material of the coating layer allowing a substrate surface to
have a surface potential.
(Detection Method)
[0079] In a detection system according to the present invention,
the presence or concentration of a target substance in a sample
solution is determined by detecting the presence or number of a
magnetic label present in the vicinity of the surface of a
detecting part. For determining the target-substance concentration
in a sample solution, preferably, the number of a magnetic label
present in the vicinity of a detecting part is determined and the
target-substance concentration is determined base on a calibration
curve previously prepared. Examples of the detection methods will
be described below.
(First Case)
[0080] A primary capturing body is immobilized on the surface of a
detecting part. Then, a sample solution containing a target
substance is brought into contact with the detecting part. On this
occasion, if the sample solution contains a desired target
substance, the primary capturing body captures the target
substance. The sample solution is washed out from the surface of
the detecting part to remove unwanted materials. Then, a solution
containing a magnetic label provided with a secondary capturing
body for capturing the target substance on the surface thereof is
brought into contact with the washed surface of the detecting part
of the detecting element (refer to FIG. 2 mentioned above).
[0081] Then, the surface of the detecting part is washed to remove
the magnetic label not bound to the detecting part. After that, the
target substance can be indirectly detected by detecting the
magnetic label. Furthermore, when the target substance captured by
the primary capturing body on the surface of the detecting part is
further captured by the secondary capturing body of the magnetic
label, the surface potential of the magnetic label and the surface
potential of the detecting element are controlled to satisfy the
above-described characteristic in the relationship
therebetween.
[0082] As an example of such a system, a case that the target
substance is an antigen, the primary capturing body is a primary
antibody, and the secondary capturing body is a secondary antibody
is cited. In this case, the capture of the target substance by the
primary capturing body is an antigen-antibody reaction. Further,
the secondary antibody here is an antibody which captures the
antigen captured by the primary antibody at a region other than the
region of the antigen where the primary antibody captured. The
primary antibody and the secondary antibody may be the same type or
different type. In addition, the antigen region captured by the
primary antibody and the antigen region captured by the secondary
antibody may be different epitopes or the same epitope.
(Second Case)
[0083] As in the First Case, a primary capturing body is
immobilized on the surface of a detecting part. Then, a target
substance is immobilized to the surface of a magnetic label. A
sample solution containing the magnetic label immobilizing the
target substance is brought into contact with the surface of the
detecting part. The surface of the detecting part is washed to
remove the magnetic label not bound to the detecting part. Then,
the target substance is indirectly detected by detecting the
magnetic label (refer to FIG. 3 mentioned above).
[0084] Furthermore, when the target substance borne by the magnetic
label is captured by the primary capturing body borne by the
surface of the detecting part, the surface potential of the
magnetic label and the surface potential of the detecting element
are controlled to satisfy the above-described characteristic in the
relationship therebetween.
[0085] As examples of such a system, cases that the target
substance is an antigen and the primary capturing body is an
antibody and that the target substance is an antibody and the
primary capturing body is an antigen are cited.
(Third Case)
[0086] A secondary capturing body is immobilized on the surface of
a magnetic label, and then a target substance is captured by the
secondary capturing body. Then, as in the First Case, a primary
capturing body is immobilized on the surface of a detecting part. A
sample solution containing the magnetic label provided with a
complex of the target substance and the secondary capturing body on
the surface thereof is brought into contact with the surface of the
detecting part so that the target substance captured by the
secondary capturing body is captured by the primary capturing body.
Then, the surface of the detecting part is washed to remove the
magnetic label not bound to the detecting part. The target
substance can be indirectly detected by detecting the magnetic
label (refer to FIG. 4 mentioned above).
[0087] Furthermore, when the target substance captured by the
secondary capturing body borne by the surface of the magnetic label
is further captured by the primary capturing body borne by the
surface of the detecting part, the surface potential of the
magnetic label and the surface potential of the detecting element
are controlled to satisfy the above-described characteristic in the
relationship therebetween.
[0088] Here, in the present invention, the state in that the
secondary capturing body borne by the surface of the magnetic label
is capturing the target substance is defined that the magnetic
label is provided with the secondary capturing body and the target
substance on the surface thereof and is included in the concept of
"magnetic label is provided with a target substance".
[0089] Further, just after the contact of a sample solution with a
detecting part, a solution containing a magnetic label provided
with a secondary capturing body may be brought into contact with
the detecting part, or both may be carried out simultaneously. In
such a case, it is thought that both reactions in the First Case
and the Third Case occur. Therefore, when the target substance on
the magnetic label is captured by the primary capturing body of the
detecting part and when the target substance captured by the
primary capturing body of the detecting part is captured by the
secondary capturing body of the magnetic label, the surface
potential of the magnetic label and the surface potential of the
detecting element are controlled to satisfy the above-described
characteristic in the relationship therebetween. Since the term
"OR" means a concept including "AND", the above-mentioned case is
within the scope of the present invention.
[0090] In the First to Third Cases, any detection methods can be
used as long as a magnetic label present in the vicinity of the
surface of a detecting part is detected. In particular, a method
using a magnetic-field effect caused by the magnetic label present
at the surface of the detecting part is preferable. Specifically, a
magnetoresistive element, a hall effect element, or a
superconducting quantum interference device can be suitably
used.
[0091] The relationships among the surface potentials of a magnetic
label, a detecting part, and a non-detecting part will be described
with reference to First to Fourth Embodiments.
First Embodiment
[0092] In this Embodiment, the surface potential .psi..sub.1 of a
magnetic label, the surface potential .psi..sub.2 of a detecting
part, and the surface potential .psi..sub.3 of a non-detecting part
are in the relationship i); and a target substance on the magnetic
label is captured by a primary capturing body of the detecting
part, or a target substance captured by a primary capturing body of
the detecting part is captured by a secondary capturing body of the
magnetic label,
.psi..sub.1.psi..sub.3>0 and .psi..sub.2=0. i)
[0093] The .psi..sub.1, .psi..sub.2, and .psi..sub.3 satisfying
this relationship are as follows:
.psi..sub.1<0, .psi..sub.2=0, and .psi..sub.3<0, or I)
.psi..sub.1>0, .psi..sub.2=0, and .psi..sub.3>0. II)
[0094] In this embodiment, since the surface potential .psi..sub.1
of the magnetic label and the surface potential .psi..sub.3 of the
non-detecting part have the same polarity, a repulsive force
(electrostatic repulsive force) is generated between the magnetic
label and the non-detecting part. In addition, the detecting part
does not exhibit a polarity. Therefore, the magnetic label is
indirectly led to the detecting part by the repulsive force between
the .psi..sub.1 and .psi..sub.3. Consequently, the non-specific
adsorption of the magnetic label to the non-detecting part is
decreased (a reduction in noise) and the detection of the target
substance can be carried out with high sensitivity and high
accuracy.
Second Embodiment
[0095] In this Embodiment, the surface potential .psi..sub.1 of a
magnetic label, the surface potential .psi..sub.2 of a detecting
part, and the surface potential .psi..sub.3 of a non-detecting part
are in the relationship ii); and a target substance borne by the
magnetic label is captured by a primary capturing body borne by the
detecting part, or a target substance captured by a primary
capturing body borne by the detecting part is captured by a
secondary capturing body borne by the magnetic label,
.psi..sub.1.psi..sub.2<0 and .psi..sub.3=0. ii)
[0096] The .psi..sub.1, .psi..sub.2, and .psi..sub.3 satisfying
this relationship are as follows:
.psi..sub.1<0, .psi..sub.2>0, and .psi..sub.3=0, or III)
.psi..sub.1>0, .psi..sub.2<0, and .psi..sub.3=0. IV)
[0097] In this embodiment, since the surface potential .psi..sub.1
of the magnetic label and the surface potential .psi..sub.2 of the
detecting part have opposite polarities, an electrostatic
attractive force is generated between the magnetic label and the
detecting part. On the other hand, since the non-detecting part
does not have a charge, an electrostatic attractive force is not
generated between the magnetic label and the non-detecting part.
Therefore, the magnetic label is led to the detecting part by the
electrostatic attractive force between the .psi..sub.1 and
.psi..sub.2. Consequently, the non-specific adsorption of the
magnetic label to the non-detecting part is decreased and the
magnetic label is rapidly and selectively led to the detecting
part. As a result, the detection of the target substance can be
carried out with high sensitivity and high accuracy.
Third Embodiment
[0098] In this Embodiment, the surface potential .psi..sub.1 of a
magnetic label, the surface potential .psi..sub.2 of a detecting
part, and the surface potential .psi..sub.3 of a non-detecting part
are in the relationship iii); and a target substance borne by the
magnetic label is captured by a primary capturing body borne by the
detecting part, or a target substance captured by a primary
capturing body borne by the detecting part is captured by a
secondary capturing body borne by the magnetic label,
.psi..sub.1.psi..sub.2<0, .psi..sub.2.psi..sub.3>0, and
|.psi..sub.2|>|.psi..sub.3|. iii)
[0099] The .psi..sub.1, .psi..sub.2, and .psi..sub.3 satisfying
this relationship are as follows:
.psi..sub.1>0 and .psi..sub.2<.psi..sub.3<0, or V)
.psi..sub.1<0 and .psi..sub.2>.psi..sub.3>0. VI)
[0100] In this embodiment, since the surface potential .psi..sub.1
of the magnetic label and the surface potential .psi..sub.2 of the
detecting part have opposite polarities, an electrostatic
attractive force is generated between the magnetic label and the
detecting part. In addition, since the surface potential
.psi..sub.1 of the magnetic label and the surface potential
.psi..sub.3 of the non-detecting part also have opposite
polarities, an electrostatic attractive force is generated between
the magnetic label and the non-detecting part. However, there is a
relationship of |.psi..sub.2|>|.psi..sub.3|, so that the
electrostatic attractive force generated between the magnetic label
and the detecting part is greater than that between the magnetic
label and the non-detecting part. Therefore, the magnetic label is
rapidly and selectively led to the detecting part, and the
detection of the target substance can be carried out with high
sensitivity and high accuracy.
Fourth Embodiment
[0101] In this Embodiment, the surface potential .psi..sub.1 of a
magnetic label, the surface potential .psi..sub.2 of a detecting
part, and the surface potential .psi..sub.3 of a non-detecting part
are in the relationship iv); and a target substance borne by the
magnetic label is captured by a primary capturing body borne by the
detecting part, or a target substance captured by a primary
capturing body borne by the detecting part is captured by a
secondary capturing body borne by the magnetic label,
.psi..sub.1.psi..sub.2<0 and .psi..sub.2.psi..sub.3<0.
iv)
[0102] The .psi..sub.1, .psi..sub.2, and .psi..sub.3 satisfying
this relationship are as follows:
.psi..sub.1>0, .psi..sub.2<0, and .psi..sub.3>0, or
VII)
.psi..sub.1<0, .psi..sub.2>0, and .psi..sub.3<0. VIII)
[0103] In this embodiment, since the surface potential .psi..sub.1
of the magnetic label and the surface potential .psi..sub.2 of the
detecting part have opposite polarities, an electrostatic
attractive force is generated between the magnetic label and the
detecting part. On the other hand, since the surface potential
.psi..sub.1 of the magnetic label and the surface potential
.psi..sub.3 of the non-detecting part have the same polarity, a
repulsive force is generated between the magnetic label and the
non-detecting part. Consequently, the non-specific adsorption of
the magnetic label to the non-detecting part is decreased and the
magnetic label is rapidly and selectively led to the detecting
part. As a result, the detection of the target substance can be
carried out with high sensitivity and high accuracy.
[0104] In this Embodiment, it is preferable that the non-detecting
part and the magnetic label each have a layered structure and that
the outermost layer of the non-detecting part and the outermost
layer (coating layer) of a magnetic structure be formed of the same
material. The magnetic structure is a magnetic label before the
immobilization of a capturing body or a target substance. By
forming the coating layers on the surfaces of the non-detecting
part and the magnetic structure with the same material, the
polarity of the surface potential of the magnetic label and the
polarity of the surface potential of the non-detecting part in a
sample solution can be readily controlled to be the same. That is,
the repulsive force can be readily generated between the magnetic
label and the non-detecting part. FIG. 6 shows a detecting element
including a non-detecting part 8 having a coating layer 16 and a
detecting part 7 and a magnetic label having a coating layer
16.
[0105] The coating layer 16 is preferably formed of a graft
polymer, which can be formed by living radical polymerization. The
living radical polymerization can precisely control the molecular
weight, molecular weight distribution (weight average molecular
weight/number average molecular weight), and graft density of the
graft polymer. By precisely controlling the graft density of the
graft polymer present in the surfaces of the non-detecting part and
the magnetic structure, the surfaces of the non-detecting part and
the magnetic label can be controlled to the same polarity and the
non-specific adsorption of the magnetic label to the non-detecting
part can be prevented. More preferably, the surfaces of the
non-detecting part and the magnetic label have the same polarity
and the absolute values of the surface zeta potentials of the
non-detecting part and the magnetic label are large. Examples of
the graft polymer include polyglycidyl methacrylate, PHEMA, and
polyethylene glycol methacrylate.
[0106] A method for forming a graft polymer will be described
below. The formation of a graft polymer on a substrate can be
carried out by at least the processes 1) and 2) below. Here, the
substrate is a non-detecting part or a magnetic structure before
the formation of a graft polymer, and the substrate surface is the
surface of the non-detecting part or the surface of the magnetic
structure before the formation of a graft polymer.
1) Process for introducing a living polymerization initiator group
to the surface of a substrate, and 2) Process for conducting living
polymerization of a monomer from the living polymerization
initiator group to form a graft polymer binding to the living
polymerization initiator group.
[0107] Generally, the living radical polymerization can freely
select the type of monomer, the degree of polymerization, and the
formation of copolymer. Therefore, the molecular weight, molecular
weight distribution, and graft density of the graft polymer on the
substrate surface can be precisely controlled. Examples of the
living polymerization include living radical polymerization, living
cation polymerization, and living anion polymerization. Among them,
the living radical polymerization is preferable because of its
simplicity of polymerization. As the living radical polymerization,
atom transfer radical polymerization, nitroxide mediated
polymerization, reversible addition fragmentation chain transfer
(RAFT) polymerization, or photoinitiated polymerization can be
used. Here, a method using the atom transfer radical polymerization
will be specifically described in detail.
Regarding the Process 1)
[0108] A method for introducing an atom transfer radical
polymerization initiator group to a substrate will be
described.
[0109] An atom transfer radical polymerization initiator group can
be introduced to the surface of a substrate by reacting a
functional group present in the surface of the substrate to a
functional group of a precursor of the atom transfer radical
polymerization initiator group. For example, the reaction can be
carried out according to the following reaction formula (I):
##STR00001##
[0110] That is, a substrate is immersed in a reaction solvent, and
then precursor 1 of an atom transfer radical polymerization
initiator group is added thereto under an inert gas atmosphere to
react a hydroxyl group of the substrate surface with a
trichlorosilyl group of precursor 1 of the atom transfer radical
polymerization initiator group. With this, the atom transfer
radical polymerization initiator group can be introduced to the
substrate surface. The functional group of precursor 1 of the atom
transfer radical polymerization initiator group may be a
trimethoxysilyl group or triethoxysilyl group instead of the
trichlorosilyl group.
[0111] As the inert gas, nitrogen gas or argon gas can be used. The
reaction solvent is not specifically limited, and examples of which
include dimethylsulfoxide, dimethylformamide, tetrahydrofuran,
benzene, toluene, and xylene.
[0112] Instead of precursor 1 of the atom transfer radical
polymerization initiator group, precursors 2 to 4 of the atom
transfer radical polymerization initiator group described below may
be used. Further, a trimethoxysilyl group or triethoxysilyl group
may be used instead of the trichlorosilyl group of precursors 2 to
4 of the atom transfer radical polymerization initiator group.
Precursor 2 of atom transfer radical polymerization initiator
group:
##STR00002##
(n: an integer of 1 to 10) Precursor 3 of atom transfer radical
polymerization initiator group:
##STR00003##
Precursor 4 of atom transfer radical polymerization initiator
group:
##STR00004##
[0113] The reaction temperature is not specifically limited as long
as the above-mentioned reaction is performed, but is preferably in
a range of from 0 up to 100.degree. C. The concentration of the
precursor of an atom transfer radical polymerization initiator
group is preferably in a range of 1 to 5 equivalents to the
concentration of the functional group of a substrate surface.
[0114] The number of the introduced living polymerization initiator
group is optionally adjusted to an intended graft density. For
example, a living polymerization initiator group can be introduced
to a substrate surface at a graft density of from 0.01
molecules/nm.sup.2 or more and 1.0 molecules/nm.sup.2 or less. In
addition, when a magnetic structure is fine magnetic particles on
which a graft polymer is formed, the average diameter of the fine
magnetic particles on which the graft polymer is formed is
preferably 3 nm or more and 500 .mu.m or less, more preferably from
5 nm or more and 1 .mu.m or less.
Regarding to the Process 2)
(Living Polymerization)
[0115] Next, atom transfer radical polymerization will be
described. In the atom transfer radical polymerization, for
example, if a copper halide-bipyridyl complex is used, a polymer
having a narrow molecular weight distribution can be obtained by
rapid transfer reaction of the polymer chain end. In this atom
transfer radical polymerization, the reaction is performed by
reversely drawing a halogen atom from the growing end with the
copper complex. Factors for controlling the reaction in the atom
transfer radical polymerization include the types of ligand and
initiator group, catalyst concentration, reaction temperature,
reaction time, and concentration. A polymer having a narrow
molecular weight distribution can be formed by optimizing these
conditions and controlling the reaction. Therefore, a graft polymer
having a uniform chain length can be readily formed by conducting
the atom transfer radical polymerization to the substrate to which
an atom transfer radical polymerization initiator group is
introduced.
[0116] Specifically, a substrate is immersed in a reaction solvent,
and a monomer which becomes a graft polymer and a transition metal
complex are added thereto to conduct an atom transfer radical
polymerization by purging the reaction system with an inert gas.
Thus, the polymerization can be performed while maintaining a
constant graft density of a graft polymer. That is, all graft
polymers can be substantially uniformly grown on the surface of the
substrate by livingly polymerizing graft polymers.
[0117] Any reaction solvent can be used as long as living
polymerization can be performed. The solvent may be used alone or
as a combination of two or more.
[0118] The inert gas may be nitrogen gas or argon gas.
[0119] The transition metal complex may be a complex consisting of
a metal halide and a ligand. The metal of the metal halide is
preferably selected from transition metal elements from Ti of
atomic number 22 to Zn of atomic number 30 and is more preferably
Fe, Co, Ni, or Cu. Among them, cuprous chloride and cuprous bromide
are most preferable.
[0120] The ligand is not specifically limited as long as coordinate
bond with a metal halide is possible. Examples of the ligand
include 2,2'-bipyridyl, 4,4'-di-(n-heptyl)-2,2'-bipyridyl,
2-(N-pentyliminomethyl)pyridine, (-)-sparteine,
tris(2-dimethylaminoethyl)amine, ethylene diamine,
dimethylglyoxime,
1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane,
1,10-phenanthroline, N,N,N',N'',N''-pentamethyldiethylene triamine,
and hexamethyl(2-aminoethyl)amine.
[0121] The transition metal complex is added to a monomer which
becomes a graft polymer at a ratio of preferably 0.001 mass % or
more and 10 mass % or less, more preferably 0.05 mass % or more and
5 mass % or less.
[0122] The polymerization temperature is preferably in a range of
from 40.degree. C. up to 100.degree. C., more preferably a range of
from 50.degree. C. up to 80.degree. C.
[0123] In addition, on the occasion of the polymerization, a free
polymerization initiator which is not immobilized to a substrate is
preferably added. The free polymer generated from this free
polymerization initiator can be used as an indicator of the
molecular weight and the molecular weight distribution of a graft
polymer formed on the substrate surface.
[0124] The free polymerization initiator is preferably the same
type as the atom transfer radical polymerization group immobilized
on the substrate. That is, when a polymerization initiator group is
introduced to a substrate by precursor 1 of an atom transfer
radical polymerization initiator group, a preferable free
polymerization initiator is ethyl 2-bromoisobutyrate. When a
polymerization initiator group is introduced to a substrate by
precursor 2 of an atom transfer radical polymerization initiator
group, a preferable free polymerization initiator is ethyl
2-bromopropionate. When a polymerization initiator group is
introduced to a substrate by precursor 3 of an atom transfer
radical polymerization initiator group, a preferable free
polymerization initiator is benzenesulfonyl chloride. When a
polymerization initiator group is introduced to a substrate by
precursor 4 of an atom transfer radical polymerization initiator
group, a preferable free polymerization initiator is benzyl
chloride.
(Graft Polymer)
[0125] The graft polymer which is formed on a substrate will be
described. The graft polymer which is formed on a substrate can be
formed by livingly polymerizing a monomer in the presence of the
substrate having an introduced living polymerization initiator
group. The graft polymer on this occasion is bound to the living
polymerization initiator group and is a non-cross-linked polymer
chain grown into a straight-chain. The graft density of the graft
polymer formed on a substrate is preferably in a range of 0.01 to 1
molecules/nm.sup.2. The graft density corresponds to a density of a
living polymerization initiator group on the substrate surface
where the graft polymer is formed and can be controlled by the
introduction ratio of the living radical polymerization initiator
group.
[0126] The graft density of a graft polymer can be determined from
the film thickness and the weight of the graft polymer formed on a
substrate. The thickness of a graft polymer formed on a substrate
can be determined by spectroscopic ellipsometry, and the weight of
a graft polymer can be determined with a precise weight scale from
a difference in the weight of a substrate before and after the
living polymerization. In such a case, the values are preferably
determined using a detecting element.
[0127] The graft polymer formed on the substrate surface is
preferably a hydrophilic polymer. Here, in the present invention,
the term "hydrophilic" means that the contact angle with water is
in a range of 0 to 90 degrees. The graft polymer has an affinity to
water and is elongated thereby in a water-soluble sample solution.
Furthermore, if the graft density of a graft polymer becomes
higher, the polymer has a structure standing perpendicular to the
substrate, like a brush. With such a structure, the blocking effect
of the graft polymer is also increased, in addition to the
repulsive force generated by that the magnetic label and the
non-detecting part have the same polarity. Examples of the blocking
polymer include 2-hydroxyethyl acrylate polymers, 2-hydroxyethyl
methacrylate polymers, acrylamide polymers, methacrylamide
polymers, polyethylene glycol acrylate polymers,
methoxypolyethylene glycol acrylate polymers, polyethylene glycol
methacrylate polymers, and methoxypolyethylene glycol methacrylate
polymers. These polymers may be used alone or as a combination of
two or more thereof.
[0128] The monomer for forming these graft polymers is used at an
amount necessary for forming a graft polymer having a desired
number average molecular weight. For example, a monomer of from 5
molecules or more and 10000 molecules or less to one living
polymerization initiator group can be used.
[0129] The number average molecular weight of a graft polymer is
preferably in a range of from 500 up to 1,000,000, more preferably
from 1,000 up to 500,000. When the number average molecular weight
of a graft polymer is lower than 500, the blocking effect may be
insufficient. On the other hand, when the number average molecular
weight of a graft polymer is higher than 1,000,000, the solubility
to water may be decreased.
[0130] Further, it is preferable that the molecular weight
distribution of a graft polymer formed on a substrate surface be
narrow for suppressing a variation in the surface potential of the
non-detecting part or the magnetic label. The molecular weight
distribution (weight average molecular weight/number average
molecular weight) of a graft polymer is preferably a 1.8 or less,
more preferably 1.5 or less.
[0131] In addition, the number average molecular weight and the
molecular weight distribution of a graft polymer formed on a
substrate surface can be estimated to be the same as those of a
free polymer generated from a free polymerization initiator as
described above. The number average molecular weight and the
molecular weight distribution of a graft polymer can be measured
with GPC (AS-8020 manufactured by Toso, eluent: water, standard
polymer: polyethylene oxide). In addition, the chain length (number
average molecular weight) and the molecular weight distribution of
a graft polymer can be controlled by adjusting the amount of
monomer, polymerization time or the like.
[0132] Further, the immobilization of a capturing body (secondary
capturing body) for capturing a target substance or the
immobilization of a target substance to a magnetic structure having
a graft polymer on the surface thereof can be carried out by the
following processes. Here, for simplifying the description, an
example for immobilizing a secondary capturing body on a magnetic
structure surface will be described, but a target substance can be
immobilized to a magnetic structure by the same manner. [0133]
Process for introducing an active group to ends of parts of the
graft polymer, and [0134] Process for binding a secondary capturing
body to the introduced active group.
[0135] The respective processes will be described in detail.
[0136] (Introduction of Active Group into the End of Graft
Polymer)
[0137] Any active group may be introduced to a graft polymer as
long as the group can be bound to a secondary capturing body.
Examples of the active group include a carboxyl group and an amino
group which can bind with a protein by an amide bond. Here, the
term "active group" means a functional group which can react with a
functional group of a secondary capturing body. The introduction of
an active group to an end of a polymer chain can be carried out,
for example, by a method for adding a chain transfer agent during a
process of polymerizing a graft polymer. The chain transfer agent
is a material which transfers the active site, generally, in
radical polymerization reaction by a chain transfer reaction. The
chain transfer agent is used for converting a polymer end to a
desired functional group.
[0138] A preferable chain transfer agent is a thiol compound. As a
thiol compound which is effective as a chain transfer agent, one
having a thiol group at one end of an alkyl chain having two or
more carbon atoms and a desired functional group at the other end
is used. The desired functional group is one for immobilizing a
secondary capturing body, namely, an active group. Examples of the
active group include a carboxyl group, an active ester group, and
an amino group. Examples of the chain transfer agent having an
active group include a mercaptoacetic acid.
[0139] Further, the amount of an active group introduced to an end
of a polymer chain can be controlled by adding both a chain
transfer agent having the active group and a chain transfer agent
having an inactive group at a desired ratio. A preferable chain
transfer agent having an inactive group is a compound having a
thiol group at one end of an alkyl chain having two or more carbon
atoms and a hydroxyl group at the other end. The ratio of the chain
transfer agent having an active group and the chain transfer agent
having an inactive group is, for example, in a range of from
1/100,000 up to 100/1 as a molar ratio of a chain transfer agent
having an active group to a chain transfer agent having an inactive
group.
[0140] After the completion of polymerization, the produced fine
magnetic particles are separated and purified by proper methods,
such as washing, filtration, decantation, precipitation
fractionation, and centrifugation, to obtain a magnetic structure
binding to a graft polymer containing an active group at one end
thereof.
[0141] (Process for Binding Secondary Capturing Body to Active
Group)
[0142] The process for binding a secondary capturing body to an
active group of a magnetic structure can be carried out by a
binding method using an amide bond as described above, for example.
Examples of the binding method using an amide bond are as follows:
[0143] introducing a carboxyl group to an end of a graft polymer
and binding the carboxyl group to an amino group of a secondary
capturing body with an amide bond, or [0144] introducing an amino
group to an end of a graft polymer and binding the amino group to a
carboxyl group of a secondary capturing body with an amide bond.
The reaction conditions for forming an amide bond, e.g., the pH and
reaction temperature, may be optionally determined depending on the
combination.
[0145] By conducting at least these processes, as shown in FIG. 10,
a magnetic label 9 having a magnetic structure and a secondary
capturing body 14 which is bound to an end of a graft polymer of
the magnetic structure through an active group 13 can be obtained.
Here, the magnetic structure includes a substrate 10, a living
polymerization initiator group 12 borne by the substrate surface,
and a graft polymer (polymer chain) 11 formed on the surface of the
substrate 10 through the living polymerization initiator group
12.
EXAMPLES
[0146] The present invention will now be described in further
detail with reference to Examples, but is not limited thereto. The
materials, composition conditions, and reaction conditions can be
freely modified within a scope of the present invention to obtain a
detecting element or a detection kit achieving similar functions
and effects.
[0147] The present invention will now be described in further
detail by referring to Examples regarding the detection methods
according to the First to Fourth Embodiments of the present
invention.
Example 1
[0148] In this Example, an example according to the First
Embodiment of the present invention will be described.
[0149] In this Example, PSA is detected by using a detecting
element including a non-detecting part having a coating layer on
the surface thereof and a detecting part having a primary antibody
for capturing PSA on the surface thereof as a combination with a
magnetic label including magnetite having a secondary antibody for
capturing PSA on the surface thereof. In this method, the detecting
part is applied with a surface potential by an external power
supply. Further, the detecting element is a magnetoresistive
element.
(1) Preparation of Magnetic Label
[0150] First, a magnetic label 9 having a secondary antibody for
capturing PSA as a secondary capturing body 4 is prepared.
Magnetite particles (average particle diameter: 50 nm) are heated
under a dry N.sub.2 atmosphere and are then dispersed in anhydrous
toluene. To this magnetite particle/toluene dispersion liquid,
aminopropyltrimethoxysilane as a silane coupling agent is added for
introducing an amino group to the surfaces of the magnetite
particles. In this regard, however, if the amino group is
excessively introduced to the magnetite particles, the isoelectric
point as magnetite is largely changed. Therefore, the amount of an
amino group to be introduced is properly controlled by pretreatment
(such as drying conditions) of the magnetite particles and
conditions (such as concentration and mixing ratio) in
silane-coupling treatment. Then, a secondary antibody, as a
secondary capturing body, for capturing PSA is immobilized by
chemically binding the amino group and a peptide chain by using a
cross-linking agent, such as glutaraldehyde, for immobilizing the
secondary antibody.
[0151] By the procedure above, a magnetic label provided with a
secondary capturing body can be obtained. Since this magnetic label
is composed of magnetite having an isoelectric point of about 6.5,
the magnetic label is positively charged in an aqueous solution
having a pH lower than this value (the acid side than 6.5).
(2) Preparation of Detecting Element
[0152] Next, a detecting element including a detecting part and a
non-detecting part is prepared. The non-detecting part includes a
substrate and a coating layer. The detecting part includes a
primary antibody, as a primary capturing body, for capturing PSA on
the surface thereof. In this Example, a magnetoresistive element
shown in FIG. 11 but the upper electrode is not yet formed is used
as a substrate 6 in FIG. 5. An Au film is formed on the surface of
the substrate 6, and a SiO.sub.2 film is formed at a region other
than a region where becomes to a detecting part. That is, a
substrate was formed by forming an Au film as an upper electrode at
a region which is used as a detecting part of the substrate for
providing a part of the detecting part and forming a SiO.sub.2 film
at a region which is used as a non-detecting part for providing a
part of the non-detecting part.
[0153] Then, a part of the non-detecting part having the SiO.sub.2
film on the surface thereof is used as a substrate and a coating
layer is formed on the surface of the substrate. First, the
SiO.sub.2 film is immersed in anhydrous toluene, and
2-(4-chloromethylphenyl)ethyltrimethoxysilane as a silane-coupling
agent is added thereto to introduce a chloromethyl group to the
surface of the SiO.sub.2 film. The progress of this reaction can be
confirmed by XPS using Cl atoms as an indicator. Then, the
SiO.sub.2 film to which the chloromethyl group is introduced is
immersed in water. Sodium dithiocarbamate is added thereto to react
with the chloromethyl group. Thus, a polymerization-initiating
point for UV graft polymerization is introduced to the surface of
the SiO.sub.2 film. The progress of this reaction can be confirmed
by XPS using N atoms and S atoms as indicators. Then, the SiO.sub.2
film is immersed in acetone, and glycydil methacrylate is added
thereto. The reaction vessel is purged with nitrogen. Then, UV
graft polymerization is performed with UV light of a wavelength of
312 to 577 nm irradiated by a UV lamp at room temperature for 2 hr
to form a polyglycidyl methacrylate on the SiO.sub.2 film surface.
Then, the SiO.sub.2 film is immersed in ammonium water adjusted to
a pH of 11 and heated. With this reaction, an amino group is
introduced to the polyglycidyl methacrylate on the SiO.sub.2 film
surface. The reaction can be confirmed by XPS by using N atoms as
an indicator.
[0154] With the above described procedures, a non-detecting part 8
having a coating layer 16 on the surface thereof can be obtained.
Here, the coating layer is a hydrophilic layer formed of a graft
layer of polyglycidyl methacrylate and therefore is excellent in
prevention of non-specific adsorption of many kinds of proteins and
is readily positively charged in an aqueous solution because of its
amino group.
[0155] Then, a primary antibody, as a primary capturing body 3, for
capturing PSA is immobilized on the surface of the Au film which is
a region serving as a detecting part. First, an ethanol solution of
10-carboxy-1-decanethiol is applied to the surface of the Au film.
With this procedure, a carboxyl group is immobilized on the Au film
surface. Then, N-hydroxysulfosuccinimide aqueous solution and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous
solution are similarly applied to the Au film surface. With these
procedures, the carboxyl group immobilized on the Au film surface
is transformed into succinimide. The primary antibody, as the
primary capturing body, for capturing PSA can be immobilized by
reacting the succinimide group with the amino group of the primary
capturing body. Further, the unreacted succinimide group on the Au
film surface may be removed by the addition of hydroxylamine
hydrochloride.
[0156] With the above-described procedures, a detecting element
including a non-detecting part 8 having a coating layer 16 of
polyglycidyl methacrylate and a detecting part 7 having a primary
antibody, as a primary capturing body 3, for capturing PSA on the
surface thereof can be prepared.
[0157] In FIG. 11, the reference numeral 18 denotes a
magnetoresistive element, 19 upper electrode, 20 free layer, 21
tunnel barrier film, 22 pin layer, and 23 lower electrode.
(3) Detection of PSA
[0158] The detection of PSA, which is known as a prostate cancer
marker, can be performed by the following processes using the
magnetic label and the detecting element prepared in the above (1)
and (2).
(a) a phosphate buffer (pH 7.0) containing an antigen (target
substance), i.e., PSA, is brought into contact with a detecting
part of the detecting element; (b) unreacted PSA is washed out with
a phosphate buffer; (c) a phosphate buffer (pH 5.5) containing a
magnetic label is brought into contact with the detecting part of
the detecting element after the completion of the processes (a) and
(b), and a current or voltage is applied to the detecting part by
an external power supply 15 connected to the detecting part 7 as
shown in FIG. 8 so that the surface potential of the detecting part
surface becomes zero; and (d) unreacted magnetic label is washed
out with a phosphate buffer and the application of the voltage or
current by the external power supply is stopped.
[0159] With these procedures, PSA as a target substance is captured
by the primary antibody to PSA and the secondary antibody to PSA
wherein the primary and secondary antibodies are present on the
surfaces of the detecting part and the magnetic label,
respectively, so that the magnetic label is immobilized in the
vicinity of the detecting part of the detecting element, as shown
in FIG. 5.
[0160] When the target substance is not present in a sample
solution, the magnetic label does not stay in the vicinity of the
detecting part of the detecting element. Therefore, the target
substance can be detected by detecting the presence or absence of
the magnetic label. Further, by previously preparing a calibration
curve showing a relationship between the magnetic label and the
number of the target substance, the amount of the target substance
contained in a sample solution can be indirectly determined based
on the number of the magnetic label.
[0161] Further, in the process (c) of this Example, since the pH of
the used buffer is 5.5 which is acidic than the isoelectric point
of magnetite, the magnetic label is positively charged. In
addition, the non-detecting part has polyglycidyl methacrylate
containing a large number of amino groups on the surface thereof
and thereby is positively charged in an aqueous solution of a pH of
about 10 or less. Therefore, the surface of the non-detecting part
is also positively charged. Furthermore, the surface of the
detecting part is adjusted by an external power supply so as to be
zero in surface potential. Therefore, a repulsive force is
generated between the magnetic label and the non-detecting part.
Consequently, the detection efficiency is improved. Here, in FIG.
8, since an insulating film of SiO.sub.2 film is formed on the
surface of a region serving as the non-detecting part, a surface
potential is imparted to the surface of the detecting part 7 only
by applying a voltage or current to the substrate. In addition, the
surface potential of only the detecting part 7 can be controlled by
forming an electrode at an area which opposes the detecting part 7
with the substrate 1 therebetween and applying a current or
voltage.
Example 2
[0162] In this Example, an example different from Example 1
according to the First Embodiment will be described.
[0163] In this Example, PSA is detected by using a detecting
element including a non-detecting part having a coating layer and a
detecting part having a primary antibody capturing PSA as a
combination with a magnetic label including a coating layer and a
magnetite provided with a secondary antibody for capturing PSA.
This Example is the same as Example 1 except that the magnetic
label has a coating layer.
(1) Preparation of Magnetic Label
[0164] First, a magnetic structure 2 having a coating layer 16 on
the surface thereof is prepared. Magnetite particles are heated
under a dry N.sub.2 atmosphere and are then dispersed in anhydrous
toluene. To this magnetite particle/toluene dispersion liquid,
2-(4-chloromethylphenyl)ethyltrimethoxysilane as a silane-coupling
agent is added for introducing a chloromethyl group to the
magnetite particles. This reaction can be confirmed by detecting Cl
atoms by XPS. The magnetite particles to which chloromethyl group
is introduced are dispersed in water. Sodium dithiocarbamate is
added thereto for the reaction with the chloromethyl group. Thus,
an initiating point for UV graft polymerization is introduced to
the surfaces of the magnetite particles. This reaction can be
confirmed by detecting N atoms and S atoms by XPS. Then, the
magnetite particles and acetone are weighed into a reaction vessel
and the magnetite particles are dispersed in the acetone by
sonication. Then, glycydil methacrylate is weighed into the
reaction vessel, and the reaction vessel is purged with nitrogen.
Then, UV graft polymerization is performed with UV light of a
wavelength of 312 to 577 nm irradiated by a UV lamp at room
temperature for 2 hr to form polyglycidyl methacrylate on the
surfaces of the magnetite particles. This reaction can be confirmed
by an increase in the particle diameters measured by a dynamic
light scattering method.
[0165] Then, a secondary antibody 4 for capturing PSA is
immobilized to a magnetic structure 2 having the coating layer 16
on the surface thereof. First, the magnetic structure is dispersed
in water, and aminoethanethiol and dithiothreitol are added
thereto. The resulting mixture is adjusted to a pH of 5 with a
hydrochloride aqueous solution or a sodium hydroxide aqueous
solution for reaction. With this, an amino group and a thiol group
are introduced to the surface of the coating layer of the magnetic
structure. Then, N-succinimidyl 3-(2-pyridyldithio)propionate is
added to the mixture and reacted at room temperature for 5 hr to
introduce a succinimide group to the magnetic structure surface via
the thiol group. The succinimide group is reacted with an amino
group of an antibody to immobilize the secondary antibody for
capturing PSA a secondary capture component.
[0166] With the above-described procedures, a magnetic label
including a coating layer of polyglycidyl methacrylate and
magnetite provided with a secondary antibody for capturing PSA can
be prepared.
[0167] In addition, the coating layer is a hydrophilic layer formed
of a graft layer of polyglycidyl methacrylate and therefore is
excellent in prevention of non-specific adsorption of many kinds of
proteins and is readily positively charged in an aqueous solution
because of its amino group.
(2) Preparation of Detecting Element
[0168] A detecting element having a detecting part and a
non-detecting part, wherein the non-detecting part is provided with
a non-detecting part coating layer and the detecting part is
provided with a primary antibody for capturing PSA as a primary
capturing body, is prepared by the same method as that in Example
1.
(3) Detection of PSA
[0169] The detection of PSA, which is known as a prostate cancer
marker, can be performed by using the magnetic label and the
detecting element according to the following processes. In this
Example, the detecting part is connected to an external power
supply for controlling the surface potential of the detecting part
as in Example 1.
(a) a phosphate buffer (pH 7.0) containing an antigen (target
substance), i.e., PSA, is brought into contact with a detecting
part of the detecting element; (b) unreacted PSA is washed out with
a phosphate buffer; (c) a phosphate buffer (pH 5.5) containing a
magnetic label is brought into contact with the detecting part of
the detecting element after the completion of the processes (a) and
(b), and a current or voltage is applied to the detecting part by
an external power supply 15 connected to the detecting part 7 as
shown in FIG. 9 so that the surface potential of the detecting part
surface becomes zero; and (d) unreacted magnetic label is washed
out with a phosphate buffer and the application of the voltage or
current by the external power supply is stopped.
[0170] With these procedures, PSA as a target substance is captured
by the primary antibody to PSA and the secondary antibody to PSA
wherein the primary and secondary antibodies are present on the
surfaces of the detecting part and the magnetic label,
respectively, so that the magnetic label 9 is immobilized in the
vicinity of the detecting part 7 of the detecting element as shown
in FIG. 6.
[0171] In the process (c) of this Example, since the pH of the used
buffer is 5.5 and the magnetic label and the non-detecting part
have polyglycidyl methacrylate containing a large number of amino
groups on the surfaces thereof, the magnetic label and the
non-detecting part are positively charged. Further, the surface
potential of the detecting part surface is controlled to zero by an
external power supply. With this, a repulsive force is generated
between the non-detecting part and the magnetic label.
Consequently, the detection efficiency can be improved.
[0172] In addition, in this Example, since the magnetic label and
the non-detecting part are provided with the respective coating
layers which are formed of substantially the same material, the
polarity of the surface potential of the magnetic label and the
polarity of the surface potential of the non-detecting part tend to
be the same even if a buffer having an arbitrary pH value is used
in the process (c). Thus, the effect of the present invention can
be obtained.
Example 3
[0173] In this Example, an example according to the Fourth
Embodiment of the present invention will be described.
[0174] In this Example, PSA is detected by using a detecting
element including a non-detecting part having a coating layer and a
detecting part having a primary antibody for capturing PSA and
enable to be imparted with a surface potential by an external power
supply as a combination with a magnetic label including a coating
layer and a magnetite provided with a secondary antibody for
capturing PSA. This Example is the same as Example 2 except that a
negative surface potential is imparted to the detecting part by an
external power supply.
(1) Preparation of Magnetic Label
[0175] A magnetic label is prepared by the same method as that in
Example 2.
(2) Preparation of Detecting Element
[0176] A detecting part 7 provided with a primary antibody for
capturing PSA and a non-detecting part 8 having a coating layer 16
on the surface thereof are prepared by the same method as that in
Example 1.
(3) Detection of PSA
[0177] The detection of PSA, which is known as a prostate cancer
marker, can be performed by using the magnetic label and the
detecting element according to the following processes.
(a) a phosphate buffer (pH 7.0) containing an antigen (target
substance), i.e., PSA, is brought into contact with a detecting
part 7 of the detecting element; (b) unreacted PSA is washed out
with a phosphate buffer; (c) a phosphate buffer (pH 5.5) containing
a magnetic label is brought into contact with the detecting part of
the detecting element after the completion of the processes (a) and
(b), and a current or voltage is applied to the detecting part by
an external power supply 15 which is connected to the detecting
part 7 as shown in FIG. 9 so that the surface potential of the
detecting part surface becomes a negative potential; and (d)
unreacted magnetic label is washed out with a phosphate buffer and
the application of the voltage or current by the external power
supply is stopped.
[0178] With these procedures, PSA as a target substance is captured
by the primary antibody to PSA and the secondary antibody to PSA
wherein the primary and secondary antibodies are present on the
surfaces of the detecting part and the magnetic label,
respectively, so that the magnetic label is immobilized in the
vicinity of the detecting part of the detecting element as shown in
FIG. 6.
[0179] In the process (c) of this Example, since the pH of the used
buffer is 5.5 and the magnetic label and the non-detecting part
have polyglycidyl methacrylate containing a large number of amino
groups on the surfaces thereof, the magnetic label and the
non-detecting part are positively charged. Further, the surface
potential of the detecting part surface can be controlled to a
negative potential by an external power supply. With this, a
repulsive force is generated between the non-detecting part and the
magnetic label and an electrostatic attractive force is generated
between the detecting part and the magnetic label. Consequently,
the detection efficiency can be improved.
[0180] In this Example, as in Example 2, the non-detecting part and
the magnetic label are provided with the respective coating layers
which are formed of substantially the same material. Therefore, the
surface potentials of the non-detecting part and the magnetic label
can be adjusted to negative potentials and the surface potential of
the detecting part surface can be adjusted to a positive potential
by changing the pH of a buffer and the surface potential of the
detecting part.
Example 4
[0181] In this Example, an example according to the Second
Embodiment of the present invention will be described.
[0182] In this Example, PSA is detected by using a detecting
element including a non-detecting part having a coating layer and a
detecting part having a primary antibody for capturing PSA and
enable to be imparted with a surface potential by an external power
supply as a combination with a magnetic label including a coating
layer and a magnetite provided with a secondary antibody for
capturing PSA. The detecting part is imparted with a surface
potential by the external power supply. In addition, the detecting
element employs a magnetoresistive element.
(1) Preparation of Magnetic Label
[0183] A magnetic label is prepared by the same method as that in
Example 2.
(2) Preparation of Detecting Element
[0184] As in Example 1, an Au film is partially formed on a
substrate surface and a SiO.sub.2 film is formed at a region other
than a region used as the detecting part. That is, on a substrate,
an Au film is formed at a region which is used as the detecting
part and a SiO.sub.2 film is formed at a region which is used as
the non-detecting part.
[0185] In this Example, a non-detecting part provided with a
coating layer of a PHEMA layer is prepared.
[0186] First, a SiO.sub.2 film is immersed in anhydrous toluene so
that a hydroxyl group of the SiO.sub.2 film reacts with a
functional group of a precursor of an atom transfer radical
polymerization initiator group according to reaction formula (I).
Thus, the atom transfer radical polymerization initiator group is
introduced to the surface of the non-detecting part.
[0187] Then, the non-detecting part introduced with the atom
transfer radical polymerization initiator group is immersed in
methanol, and ethyl 2-bromoisobutyrate as a free polymerization
initiator is added thereto and CuBr, 2,2'-bipyridyl is further
added thereto. Oxygen in the reaction system is removed by freeze
vacuum degassing, and then the reaction system is purged with
nitrogen, followed by the atom transfer radical polymerization of
an HEMA (2-hydroxyethyl methacrylate) monomer for a predetermined
period of time. Further, the molecular weight and a molecular
weight distribution of PHEMA generated from ethyl
2-bromoisobutyrate added as a free polymerization initiator are
measured to confirm that PHEMA has a number average molecular
weight of 60,000 and a molecular weight distribution of 1.07. With
these results, the graft polymer grafted on the non-detecting part
is confirmed to be a polymer having a uniform chain length. The
film thickness and weight of the graft polymer grafted on the
non-detecting part are measured to confirm that the graft polymer
has a graft density of 0.6 molecules/nm.sup.2.
[0188] With the above-described procedures, a non-detecting part
coating layer can be formed on the surface of a non-detecting part.
The PHEMA layer is a hydrophilic layer having a large number of
hydroxyl groups and is excellent in prevention of non-specific
adsorption of many kinds of proteins.
[0189] Further, in this Example, a detecting part provided with a
primary antibody for capturing PSA is prepared by the same method
as that in Example 2.
(3) Detection of PSA
[0190] The detection of PSA, which is known as a prostate cancer
marker, can be performed by using the magnetic label and the
detecting element according to the following processes.
(a) a phosphate buffer (pH 7.0) containing an antigen (target
substance), i.e., PSA, is brought into contact with a detecting
part of the detecting element; (b) unreacted PSA is washed out with
a phosphate buffer; (c) a phosphate buffer having a pH near the
isoelectric point of PHEMA forming a coating layer of a
non-detecting part and containing a magnetic label is brought into
contact with the detecting part of the detecting element after the
completion of the processes (a) and (b), and a current or voltage
is applied to the detecting part by an external power supply 15
which is connected to the detecting part 7 as shown in FIG. 9 so
that the surface potential of the detecting part surface becomes a
negative potential; and (d) unreacted magnetic label is washed out
with a phosphate buffer and the application of the voltage or
current by the external power supply is stopped.
[0191] With these procedures, PSA as a target substance is captured
by the primary antibody to PSA and the secondary antibody to PSA
wherein the primary and secondary antibodies are present on the
surfaces of the detecting part and the magnetic label,
respectively, so that the magnetic label is immobilized in the
vicinity of the detecting part of the detecting element as shown in
FIG. 6.
[0192] In the process (c) of this Example, since a buffer having a
pH near the isoelectric point (near neutral) of PHEMA which forms a
coating layer of a non-detecting part is used, the surface of the
non-detecting part is actually electrically neutral. Further, the
surface potential of the detecting part surface can be controlled
to a negative potential by an external power supply. In addition,
the magnetic label has polyglycidyl methacrylate containing a large
number of amino groups on the surface thereof and thereby is
positively charged in a near neutral condition. With this, an
electrostatic attractive force is generated between the detecting
part and the magnetic label. Consequently, the detection efficiency
can be improved.
Example 5
[0193] In this Example, an example according to the Third
Embodiment of the present invention will be described.
[0194] In this Example, PSA is detected by using a detecting
element including a non-detecting part having a coating layer and a
detecting part having a primary antibody for capturing PSA and
enable to be imparted with a surface potential by an external power
supply as a combination with a magnetic label including magnetite
provided with a secondary antibody for capturing PSA. The detection
is carried out as in Example 2 except that the pH of a buffer
solution for a sample containing a magnetic label is adjusted to a
value higher than the isoelectric point of a magnetic structure and
that the absolute value of the surface potential of the detecting
part surface is larger than that of the non-detecting part and both
surface potentials are positive.
(1) Preparation of Magnetic Label
[0195] A magnetic label is prepared by the same method as that in
Example 1.
(2) Preparation of Detecting Element
[0196] A detecting part provided with a primary antibody for
capturing PSA and a non-detecting part provided with a
non-detecting part coating layer are prepared by the same method as
that in Example 2.
(3) Detection of PSA
[0197] The detection of PSA, which is known as a prostate cancer
marker, can be performed by using the magnetic label and the
detecting element according to the following processes.
(a) a phosphate buffer (pH 7.0) containing an antigen (target
substance), i.e., PSA, is brought into contact with a detecting
part of the detecting element; (b) unreacted PSA is washed out with
a phosphate buffer; (c) a phosphate buffer (pH 7.5) containing a
magnetic label is brought into contact with the detecting part of
the detecting element after the completion of the processes (a) and
(b), and a current or voltage is applied to the detecting part by
an external power supply 15 connected to the detecting part 7 as
shown in FIG. 9 so that the surface potential of the detecting part
surface becomes a positive potential; and (d) unreacted magnetic
label is washed out with a phosphate buffer and the application of
the voltage or current by the external power supply is stopped.
[0198] With these procedures, PSA as a target substance is captured
by the primary antibody to PSA and the secondary antibody to PSA
wherein the primary and secondary antibodies are present on the
surfaces of the detecting part and the magnetic label,
respectively, so that the magnetic label is immobilized in the
vicinity of the detecting part of the detecting element as shown in
FIG. 6.
[0199] In the process (c) of this Example, since the pH of the used
buffer is 7.5 which is basic than the isoelectric point of the
magnetite, the magnetic label is negatively charged. In addition,
since the non-detecting part has polyglycidyl methacrylate
containing a large number of amino groups on the surface thereof,
the non-detecting part is positively charged at pH 7.5. The
detecting part is also positively charged by an external power
supply. However, the detecting part is imparted with a surface
potential larger than that of the non-detecting part by the
external power supply. Therefore, the electrostatic attractive
force between the detecting part and the magnetic label is larger
than that between the non-detecting part and the magnetic label.
Consequently, the detection efficiency is improved.
[0200] While the present invention has been described with
reference to exemplary embodiments, it is to be understood that the
invention is not limited to the disclosed exemplary embodiments.
The scope of the following claims is to be accorded the broadest
interpretation so as to encompass all such modifications and
equivalent structures and functions.
[0201] This application claims the benefit of Japanese Patent
Applications No. 2006-100683, filed Mar. 31, 2006, and No.
2006-317401, filed Nov. 24, 2006, which are hereby incorporated by
reference herein in their entirety.
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