U.S. patent application number 16/089007 was filed with the patent office on 2019-05-02 for biosensor and biochip.
This patent application is currently assigned to TDK CORPORATION. The applicant listed for this patent is TDK CORPORATION. Invention is credited to Susumu HARATANI, Takashi KIKUKAWA, Sumiko KITAGAWA, Sachio TSUBOIKE, Haruki YUGA.
Application Number | 20190128882 16/089007 |
Document ID | / |
Family ID | 59964519 |
Filed Date | 2019-05-02 |
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United States Patent
Application |
20190128882 |
Kind Code |
A1 |
HARATANI; Susumu ; et
al. |
May 2, 2019 |
BIOSENSOR AND BIOCHIP
Abstract
What provided is a biosensor including: a substrate having a
surface with first and second regions adjacent to each other; a
magnetoresistance effect element that is disposed at least on the
first region and is configured for a detected resistance value to
be changed based on an input magnetic field; a protective film that
is disposed on both the first region and the second region, covers
a surface of the magnetoresistance effect element, is disposed on
the top part of the first region and contains an affinity substance
capable of recognizing the biomolecule on the outer surface of the
first region exclusively; and an adsorption prevention film that is
disposed on at least the top part of the second region and is
substantially free of the affinity substance, wherein the
protective film and the adsorption prevention film are made of
different materials.
Inventors: |
HARATANI; Susumu; (Tokyo,
JP) ; TSUBOIKE; Sachio; (Tokyo, JP) ;
KITAGAWA; Sumiko; (Tokyo, JP) ; KIKUKAWA;
Takashi; (Tokyo, JP) ; YUGA; Haruki; (Tokyo,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TDK CORPORATION |
Tokyo |
|
JP |
|
|
Assignee: |
TDK CORPORATION
Tokyo
JP
|
Family ID: |
59964519 |
Appl. No.: |
16/089007 |
Filed: |
March 24, 2017 |
PCT Filed: |
March 24, 2017 |
PCT NO: |
PCT/JP2017/012043 |
371 Date: |
September 27, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54373 20130101;
G01N 27/72 20130101; B82Y 15/00 20130101; C12Q 1/6825 20130101;
G01N 27/00 20130101; B01L 3/502715 20130101; C12Q 2565/629
20130101; G01N 27/745 20130101; G01N 2015/0065 20130101; G01N
33/543 20130101; G01R 33/093 20130101; B82Y 25/00 20130101; G01R
33/098 20130101; G01N 33/54393 20130101; G01N 15/0656 20130101;
G01R 33/1269 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 27/74 20060101 G01N027/74 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 2016 |
JP |
2016-063490 |
May 25, 2016 |
JP |
2016-104468 |
Jul 22, 2016 |
JP |
2016-144124 |
Jul 22, 2016 |
JP |
2016-144125 |
Jul 22, 2016 |
JP |
2016-144357 |
Claims
1. A biosensor for detecting a biomolecule in a sample, the
biosensor comprising: a substrate having a surface in which a first
region and a second region disposed adjacent to the first region
are formed; a magnetoresistance effect element that is disposed at
least on the first region and is configured for a detected
resistance value to be changed based on an input magnetic field; a
protective film that is disposed on both the first region and the
second region, covers a surface of the magnetoresistance effect
element, is disposed on the top part of the first region and
contains an affinity substance capable of recognizing the
biomolecule on the outer surface of the first region exclusively;
and an adsorption prevention film that is disposed on at least the
top part of the second region and is substantially free of the
affinity substance, wherein the protective film and the adsorption
prevention film are made of different materials.
2. The biosensor according to claim 1, further comprising a
substance that reduces nonspecific adsorption of the biomolecules
onto the outer surface of the adsorption prevention film.
3. The biosensor according to claim 1, wherein a material
constituting the protective film is a noble metal and a material
constituting the adsorption prevention film is an oxide.
4. The biosensor according to claim 1, wherein a material
constituting the protective film is an oxide and a material
constituting the adsorption prevention film is a noble metal.
5. The biosensor according to claim 3, wherein the noble metal is
at least one selected from the group consisting of gold, silver,
platinum, rhodium, ruthenium and palladium.
6. The biosensor according to claim 3, wherein the oxide is at
least one selected from the group consisting of alumina, silica,
titanium oxide, zirconium oxide, indium oxide, tantalum oxide, zinc
oxide, gallium oxide and tin oxide.
7. The biosensor according to claim 4, wherein the substance that
reduces nonspecific adsorption is at least one selected from the
group consisting of a thiol group, an isothiocyanate group and a
disulfide group.
8. The biosensor according to claim 3, wherein the substance that
reduces nonspecific adsorption has at least one of an alkoxysilane
group and a phosphonic acid group.
9. The biosensor according to claim 1, wherein the protective film
is composed of a plurality of films.
10. The biosensor according to claim 9, wherein the adsorption
prevention film is disposed on either of the first region and the
second region on a film other than the top film among the plurality
of films constituting the protective film.
11. The biosensor according to claim 9, wherein the adsorption
prevention film is disposed only on the second region on a film
other than the top film among the plurality of films constituting
the protective film.
12. A biochip comprising the biosensor according to claim 1.
13. The biosensor according to claim 2, wherein a material
constituting the protective film is a noble metal and a material
constituting the adsorption prevention film is an oxide.
14. The biosensor according to claim 2, wherein a material
constituting the protective film is an oxide and a material
constituting the adsorption prevention film is a noble metal.
15. The biosensor according to claim 4, wherein the noble metal is
at least one selected from the group consisting of gold, silver,
platinum, rhodium, ruthenium and palladium.
16. The biosensor according to claim 14, wherein the noble metal is
at least one selected from the group consisting of gold, silver,
platinum, rhodium, ruthenium and palladium.
17. The biosensor according to claim 4, wherein the oxide is at
least one selected from the group consisting of alumina, silica,
titanium oxide, zirconium oxide, indium oxide, tantalum oxide, zinc
oxide, gallium oxide and tin oxide.
18. The biosensor according to claim 14, wherein the oxide is at
least one selected from the group consisting of alumina, silica,
titanium oxide, zirconium oxide, indium oxide, tantalum oxide, zinc
oxide, gallium oxide and tin oxide.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to a biosensor and a
biochip.
[0002] Priority is claimed on Japanese Patent Application No.
2016-063490, filed Mar. 28, 2016, Japanese Patent Application No.
2016-104468, filed May 25, 2016, Japanese Patent Application No.
2016-144124, filed Jul. 22, 2016, Japanese Patent Application No.
2016-144125, filed Jul. 22, 2016, and Japanese Patent Application
No. 2016-144357, filed Jul. 22, 2016, the content of which is
incorporated herein by reference.
BACKGROUND ART
[0003] As a magnetic sensor, a magnetoresistance effect element
such as a giant magnetoresistance effect (GMR) element, a magnetic
tunnel junction (TMR) element, and an anisotropic magnetoresistance
effect (AMR) element is often used (for example, refer to Published
Japanese Translation No. S/H 2005-513475 of the PCT International
Publication and Japanese Unexamined Patent Application, First
Publication No. 2008-039782). A magnetoresistance effect element is
an element whose output resistance value changes according to an
input magnetic field, and it is possible to measure a change in the
detected magnetic field on the basis of the output resistance
value.
[0004] FIG. 6 and FIG. 7 are diagrams for explaining a biosensor
500 of the related art. As shown in FIG. 6, the biosensor 500
includes a substrate 101, a magnetoresistance effect element 102, a
protective film 107, and a biomolecule capturing layer 109 that
captures target biomolecules in that order. When biomolecules in a
sample are captured on the biomolecule capturing layer 109,
magnetic beads having affinity for the biomolecules are captured on
the biomolecule capturing layer 109 via the biomolecules and a
magnetic field is then horizontally applied (an applied magnetic
field 105), a stray magnetic field 111 is generated from magnetic
beads 104 and the stray magnetic field 111 is input to the
magnetoresistance effect element 102.
[0005] FIG. 7 is a diagram showing details of the magnetoresistance
element 102 of the related art used in the biosensor 500 of the
related art. As shown in FIG. 7, the magnetoresistance effect
element 102 has a meander structure with sets of three lines.
SUMMARY
Technical Problem
[0006] As shown in FIG. 7, in the meander structure, there are
cases in which magnetic beads 104 are disposed on a
magnetoresistance effect element 102 and disposed between the
magnetoresistance effect elements 102. The output varies positively
or negatively according to a difference in the disposition, that
is, a relative position between the magnetoresistance effect
element 102 and the magnetic beads 104. Therefore, when magnetic
beads are present both on thin lines and between thin lines of a
magnetoresistance effect element having a meander structure, there
are problems of measured values of a concentration varying and
sufficient accuracy not being obtained.
[0007] The present disclosure has been made in view of the above
circumstances and provides a biosensor through which a measurement
error due to magnetic beads present both on thin lines and between
thin lines of a magnetoresistance effect element having a meander
structure is avoided and it is possible to detect biomolecules in a
sample with high accuracy.
Solution to Problem
[0008] The inventors conducted extensive studies in order to
address the above problem, and as a result, found that, when an
adsorption prevention film is disposed between thin lines of a
magnetoresistance effect element, a measurement error due to
magnetic beads present both on thin lines and between thin lines of
a magnetoresistance effect element can be avoided, and thereby
completed the present disclosure.
[0009] That is, the present disclosure is directed to the following
aspect.
[0010] A biosensor according to a first aspect of the present
disclosure is a biosensor for detecting biomolecules in a sample,
the biosensor including:
[0011] a substrate having a surface in which a first region and a
second region disposed adjacent to the first region are formed;
[0012] a magnetoresistance effect element that is disposed at least
on the first region and is configured for a detected resistance
value to be changed based on an input magnetic field;
[0013] a protective film that is disposed on both the first region
and the second region, covers a surface of the magnetoresistance
effect element, is disposed on the top part of the first region and
contains an affinity substance capable of recognizing the
biomolecule on the outer surface of the first region exclusively;
and
[0014] an adsorption prevention film that is disposed on at least
the top part of the second region and is substantially free of the
affinity substance,
[0015] wherein the protective film and the adsorption prevention
film are made of different materials.
BRIEF DESCRIPTION OF DRAWINGS
[0016] FIG. 1 is a perspective view schematically showing a
biosensor according to a first embodiment of the present
disclosure.
[0017] FIG. 2 is a cross-sectional view schematically showing the
biosensor according to the first embodiment of the present
disclosure.
[0018] FIG. 3 is a cross-sectional view schematically showing a
biosensor according to a second embodiment of the present
disclosure.
[0019] FIG. 4 is a cross-sectional view schematically showing a
biosensor according to a third embodiment of the present
disclosure.
[0020] FIG. 5 is a cross-sectional view schematically showing a
biosensor according to a fourth embodiment of the present
disclosure.
[0021] FIG. 6 is a cross-sectional view of a magnetic detection
type biosensor of the related art.
[0022] FIG. 7 is a perspective view of the magnetic detection type
biosensor of the related art.
DESCRIPTION OF EMBODIMENTS
[0023] Biosensor
[0024] A biosensor according to an embodiment of the present
disclosure includes a substrate, a magnetoresistance effect
element, a protective film, and an adsorption prevention film.
[0025] The substrate has a surface in which a first area and a
second area disposed adjacent to the first area are formed.
[0026] The magnetoresistance effect element is disposed on at least
the first area and is configured such that a resistance value
detected according to an input magnetic field varies.
[0027] The protective film is disposed on both the first area and
the second area and covers a surface of the magnetoresistance
effect element, and is disposed on the top part of the first area.
In addition, the protective film contains an affinity substance
that allows recognition of the biomolecules on the outer surface
only on the first area. The adsorption prevention film is disposed
on at least the top part of the second area and is substantially
free of the affinity substance.
[0028] The protective film and the adsorption prevention film are
made of different materials.
[0029] According to the above aspects of the present invention, a
measurement error due to magnetic beads present both on thin lines
and between thin lines of the magnetoresistance effect element
having a meander structure is avoided, and it is possible to detect
biomolecules in a sample with high accuracy.
[0030] Here, in this specification, the "biosensor" refers to a
sensor that detects biological materials (that may be naturally
derived or chemically synthesized) such as enzymes, antigens,
antibodies, and nucleic acids (including not only DNA, RNA, and the
like but also artificial nucleic acids, for example, LNA).
[0031] In addition, "adsorption prevention film" refers to a film
for preventing biomolecules or magnetic beads from being adsorbed
between thin lines of the magnetoresistance effect element having a
meander structure.
[0032] When the biosensor of the present embodiment includes an
adsorption prevention film that is disposed on at least the top
part of the second area and is substantially free of the affinity
substance, a measurement error due to magnetic beads present both
on thin lines and between thin lines of the magnetoresistance
effect element having a meander structure is avoided, and it is
possible to detect biomolecules in a sample with high accuracy.
[0033] In this specification, "substantially free of an affinity
substance" refers to a case in which no affinity substance is
contained or a case in which, for example, when an affinity
substance is nonspecifically adsorbed onto an adsorption prevention
film, the affinity substance is contained only in an amount at
which it is not possible to capture biomolecules.
[0034] Structure of Biosensor
[0035] Differences in structures of biosensors of the present
embodiment will be described below with reference to the drawings.
Here, in the drawings used in the following descriptions, in order
to facilitate understanding of features of the present embodiment,
featured parts are enlarged for convenience of illustration in some
cases, and dimensional proportions and the like of components are
not necessarily the same as those of actual components.
First Embodiment
[0036] FIG. 1 is a perspective view schematically showing a
biosensor according to a first embodiment of the present
disclosure.
[0037] As shown in FIG. 1, a magnetoresistance effect element 12
has a meander structure with sets of three lines, and an adsorption
prevention film 16 is disposed between thin lines of the
magnetoresistance effect element 12.
[0038] In addition, FIG. 2 is a cross-sectional view schematically
showing the biosensor according to the first embodiment of the
present disclosure, and is a cross-sectional view of the biosensor
taken along the line X-X' in FIG. 1.
[0039] A first area, a second area, a first plane, and a second
plane, which will be described below, are introduced for
convenience in order to define a positional relationship between
members on an area or plane in a virtual area or plane. Here, on
the biosensor of the present embodiment, the first area and the
second area are alternately repeatedly provided.
[0040] The biosensor 100 of the present embodiment detects
biomolecules in a sample.
[0041] The biosensor 100 includes a substrate 11, the
magnetoresistance effect element 12, a protective film 17, and the
adsorption prevention film 16.
[0042] The substrate 11 has a surface in which a first area A and a
second area B disposed adjacent to the first area A are formed.
[0043] The magnetoresistance effect element 12 is disposed on at
least the first area A and is configured such that a resistance
value detected according to an input magnetic field varies.
[0044] The protective film 17 is disposed on both the first area A
and the second area B and covers a surface of the magnetoresistance
effect element 12 and is disposed on the top part of the first area
A. In addition, the protective film 17 contains an affinity
substance 19 (hereinafter referred to as a "first affinity
substance") that allows recognition of biomolecules on the outer
surface only on the first area A.
[0045] The adsorption prevention film 16 is disposed on at least
the top part of the second area B and is substantially free of the
affinity substance 19.
[0046] The protective film 17 and the adsorption prevention film 16
are made of different materials.
[0047] The adsorption prevention film 16 is substantially free of
the affinity substance 19, and is made of a material different from
that of a protective layer 17. Thus, it is possible to reduce
adsorption of biomolecules or magnetic beads 14 on the adsorption
prevention film. In addition, a measurement error due to magnetic
beads present both on thin lines and between thin lines of the
magnetoresistance effect element having a meander structure is
avoided, and it is possible to detect biomolecules in a sample with
high accuracy.
[0048] Here, the meaning of "at least" in the sentence of "disposed
on at least the first area" regarding the magnetoresistance effect
element 12 will be described with reference to FIG. 2. As shown in
FIG. 2, a configuration in which the adsorption prevention film 16
extends in a width direction (left-right direction of the plane of
the paper) on the entire second area B is formed. However, in such
a configuration, when the magnetoresistance effect element 12 is
disposed so that it extends not only to the first area A but also
the second area B, that is, when the magnetoresistance effect
element 12 is disposed so that it overlaps the adsorption
prevention film 16 in a plan view, this configuration is preferable
in consideration of ease of production.
[0049] In addition, in the present embodiment, the magnetic beads
14 contain a second affinity substance (not shown) that allows
recognition of a part different from the biomolecule recognition
part of the first affinity substance 19. The magnetic beads 14
accumulate on the protective film 17 via a first affinity
substance-biomolecule-second affinity substance complex. Then, when
a magnetic field is horizontally applied (an applied magnetic field
15), a stray magnetic field is generated from the magnetic beads 14
and a stray magnetic field is input to the magnetoresistance effect
element 12.
[0050] As shown in FIG. 2, the surface of the magnetoresistance
effect element 12 is covered with the protective film 17, and the
outer surface of the protective film 17 contains the first affinity
substance 19 that captures biomolecules to be detected. The
magnetic beads 14 also contain a second affinity substance (not
shown) that captures biomolecules. The first affinity substance 19
and the second affinity substance allow recognition of different
parts in biomolecules. That is, they can form a first affinity
substance-biomolecule-second affinity substance complex.
[0051] In addition, as shown in FIG. 1, the electrode terminal is
disposed on a plane which is positioned away from the main surface
of the substrate 11 and immediately above the magnetoresistance
effect element 12. The electrode terminal is connected when it is
disposed at a position in contact with the magnetoresistance effect
element 12.
Second Embodiment
[0052] FIG. 3 is a cross-sectional view schematically showing a
biosensor according to a second embodiment of the present
disclosure. Here, in the drawings following FIG. 3, components that
are the same as those shown in the drawings explained above are
denoted with the same reference numerals as in the drawings
explained above, and details thereof will not be described.
[0053] A biosensor 200 is the same as the biosensor 100 shown in
FIG. 1 except that a protective film is composed of a plurality of
films. That is, in the biosensor 200, a second protective film 20
is laminated on one surface of the substrate 11. In addition, the
magnetoresistance effect element 12 is disposed on a first plane a
in the second protective film 20. In addition, the second
protective film 20 is laminated on a second plane b which is a
surface of the magnetoresistance effect element 12. In addition,
the protective film 17 is laminated on both the first area A and
the second area B on the surface of the second protective film 20.
In addition, the adsorption prevention film 16 is laminated on the
surface of the protective film 17 on the second area B. In
addition, the surface of the protective film 17 on the first area A
contains the affinity substance 19. In other words, the second
protective film 20 and the protective film 17 are laminated on both
the first area A and the second area B on the magnetoresistance
effect element 12 in that order. In addition, the adsorption
prevention film 16 is laminated on the surface of the protective
film 17 on the second area B.
[0054] In the biosensor 200, the adsorption prevention film 16 is
substantially free of the affinity substance 19, and the adsorption
prevention film 16, the protective film 17 and the second
protective film 20 are made of different materials.
[0055] The biosensor 200 shown in FIG. 3 is used to detect
biomolecules in a sample based on the same principle as in the
biosensor 100 shown in FIG. 2.
Third Embodiment
[0056] FIG. 4 is a cross-sectional view schematically showing a
biosensor according to a third embodiment of the present
disclosure.
[0057] A biosensor 300 is the same as the biosensor 200 shown in
FIG. 3 except that an adsorption prevention film is disposed on
both the first area A and the second area B. That is, in the
biosensor 300, the second protective film 20 is laminated on one
surface of the substrate 11. In addition, the magnetoresistance
effect element 12 is disposed on the first plane a in the second
protective film 20. In addition, the second protective film 20 is
laminated on the second plane b which is a surface of the
magnetoresistance effect element 12. In addition, the adsorption
prevention film 16 is laminated on both the first area A and the
second area B on the surface of the second protective film 20. In
addition, the protective film 17 is laminated on the surface of the
adsorption prevention film 16 on the first area A and the surface
of the protective film 17 on the first area A contains the affinity
substance 19. In other words, in the first area A, the adsorption
prevention film 16 that is interposed between the second protective
film 20 and the protective film 17 is disposed.
[0058] In the biosensor 300, the adsorption prevention film is
substantially free of the affinity substance 19, and the adsorption
prevention film 16, the protective film 17 and the second
protective film 20 are made of different materials.
[0059] The biosensor 300 shown in FIG. 4 is used to detect
biomolecules in a sample based on the same principle as in the
biosensor 100 shown in FIG. 2.
Fourth Embodiment
[0060] FIG. 5 is a cross-sectional view schematically showing a
biosensor according to a fourth embodiment of the present
disclosure.
[0061] A biosensor 400 is the same as the biosensor 200 shown in
FIG. 3 except that the adsorption prevention film 16 is disposed on
the top part of the second area B and the protective film 17 is
disposed on the top part of the first area A. That is, in the
biosensor 400, the second protective film 20 is laminated on one
surface of the substrate 11. In addition, the magnetoresistance
effect element 12 is disposed on the first plane a in the second
protective film 20. In addition, the second protective film 20 is
laminated on the second plane b which is a surface of the
magnetoresistance effect element 12. In addition, the adsorption
prevention film 16 is laminated on the surface of the second
protective film 20 on the second area B. In addition, the
protective film 17 is laminated on the surface of the second
protective film 20 on the first area A. In addition, the surface of
the protective film 17 on the first area A contains the affinity
substance 19. In other words, the adsorption prevention film 16 is
disposed on the top part of the second area B, and the protective
film 17 is disposed on the top part of the first area A.
[0062] In the biosensor 400, the adsorption prevention film 16 is
substantially free of the affinity substance 19, and the adsorption
prevention film 16, the protective film 17 and the second
protective film 20 are made of different materials.
[0063] The biosensor 400 shown in FIG. 5 is used to detect
biomolecules in a sample based on the same principle as in the
biosensor 100 shown in FIG. 2.
[0064] The biosensor according to the present embodiment is not
limited to those shown in FIGS. 1 to 5, and a biosensor in which
some components of those shown in FIGS. 1 to 5 are modified or
deleted and a biosensor in which other components are additionally
added to those described above may be used as long as effects
thereof are not impaired.
[0065] For example, in the biosensors shown in FIGS. 1 to 5, the
adsorption prevention film may be disposed on the entire top part
in which there is no magnetoresistance effect element.
[0066] Components of Biosensor
[0067] Components of the biosensor of the present embodiment will
be described below in detail.
[0068] Substrate
[0069] As a material of the substrate, for example, a semiconductor
such as silicon and AlTiC or a conductor, or a material made of an
insulator such as alumina or glass may be exemplified, and a form
thereof is not particularly limited.
[0070] The thickness of the substrate is not particularly limited,
but it may be, for example, 400 .mu.m or more and 2000 .mu.m or
less. When the thickness of the substrate is in such a range, it is
possible to obtain a thin and lightweight biosensor having an
appropriate strength.
[0071] Here, "the thickness of the substrate" refers to the
thickness of the entire substrate. For example, the thickness of
the substrate composed of a plurality of layers refers to the total
thickness of all layers constituting the substrate.
[0072] Magnetoresistance Effect Element
[0073] The magnetoresistance effect element is not particularly
limited as long as it is an element that uses a phenomenon in which
a magnetic field influence is received and an electrical resistance
changes. An element of a type including a magnetization fixed layer
having a magnetization direction fixed in a certain direction in
the plane of the laminate and a magnetization free layer whose
magnetization direction changes according to an external magnetic
field is preferable. In addition, in the magnetoresistance effect
element, preferably, a magnetization fixed direction of the
magnetization fixed layer is substantially parallel or
substantially antiparallel to a direction of a magnetic field (the
applied magnetic field 15) applied for magnetic bead excitation,
and is a film surface direction of the magnetoresistance effect
element.
[0074] In the present embodiment, description including the terms
substantially parallel or substantially antiparallel, means
approximately parallel or anti-parallel, and includes deviation
within a range of 0.1.degree. or more and 10.degree. or less.
[0075] In addition, the magnetoresistance effect element includes a
magnetization fixed layer, an intermediate layer made of a
nonmagnetic conductor or an insulator, and a magnetization free
layer, and preferably includes a laminate including the
intermediate layer interposed between the magnetization fixed layer
and the magnetization free layer.
[0076] Here, when the intermediate layer is made of a nonmagnetic
conductor, the magnetoresistance effect element is generally called
a giant magnetoresistance (GMR) effect element and when the
intermediate layer is made of an insulator, the magnetoresistance
effect element is called a tunnel type magnetoresistance (TMR)
effect element. A resistance of the magnetoresistance effect
element changes according to an angle between a magnetization
direction of the magnetization fixed layer and an average
magnetization direction of the magnetization free layer. Generally,
the magnetization direction of the magnetization fixed layer is
defined as a magnetic sensing direction.
[0077] The magnetization free layer is made of, for example, a soft
magnetic film of NiFe or the like. The intermediate layer is made
of, for example, a conductor film of Cu or the like or made of an
insulator film of an alumina-magnesium oxide or the like.
[0078] The magnetization fixed layer is made of an
antiferromagnetic film and a magnetization fixed film, and the
magnetization fixed film is in contact with the intermediate layer.
The antiferromagnetic film is made of, for example, an
antiferromagnetic Mn alloy such as IrMn and PtMn. The magnetization
fixed film is made of, for example, a ferromagnetic material such
as CoFe and NiFe, or may have a configuration in which a Ru thin
film layer is interposed between CoFe layers or the like.
[0079] Magnetic Beads
[0080] Magnetic beads are not particularly limited as long as they
are magnetic particles. For example, iron oxide particles may be
exemplified. The diameter of the magnetic beads depends on the
balance with the area of the protective film. For example, the
diameter is preferably 0.01 .mu.m or more and 100 .mu.m or less,
more preferably 0.05 .mu.m or more and 50 .mu.m or less, and
particularly preferably 0.1 .mu.m or more and 5 .mu.m or less.
[0081] The magnetic beads contain a second affinity substance that
specifically binds to biomolecules, and capture biomolecules via
the second affinity substance. The magnetic beads may be magnetic
beads to which a second affinity substance is added by a coating
treatment or the like or magnetic beads composed of the second
affinity substance itself.
[0082] Preferably, the surface of the magnetic beads is coated with
a polymer or silica matrix according to biomolecules to be
captured. When ligands are desired to be captured as biomolecules,
the surface of the magnetic beads is preferably hydrophilic, and
when antibodies are desired to be captured as biomolecules, the
surface of the magnetic beads is preferably hydrophobic.
[0083] Protective Film
[0084] The protective film is not particularly limited as long as
it can protect the magnetoresistance effect element. Examples of
the material of the protective film include oxides such as alumina,
silica, titanium oxide, zirconium oxide, indium oxide, tartaric
oxide, zinc oxide, gallium oxide, and tin oxide; noble metals such
as gold, silver, platinum, rhodium, ruthenium, and palladium; and
inorganic substances such as aluminum nitride, and silicon nitride,
and organic substances such as a polyimide.
[0085] The protective film may be composed of one layer (single
layer) or a plurality of layers such as two or more layers. In
addition, when the protective film is composed of a plurality of
layers, the plurality of layers may be the same as or different
from each other, and combinations of the plurality of layers are
not particularly limited.
[0086] The thickness of the protective film is preferably 1 nm or
more and 1000 nm or less, more preferably 1 nm or more and 100 nm
or less, and particularly preferably 1 nm or more and 15 nm or
less.
[0087] Here, "the thickness of the protective film" refers to the
thickness of the entire protective film. For example, the thickness
of the protective film composed of a plurality of layers refers to
the total thickness of all layers constituting the protective
film.
[0088] The outer surface of the protective film is a surface that
comes in contact with biomolecules in a sample. The outer surface
contains a first affinity substance that specifically binds to
biomolecules to be detected. In addition, the magnetic beads also
contain a second affinity substance that specifically binds to
biomolecules.
[0089] When these affinity substances are contained, biomolecules
are fixed to the outer surface of the protective film via the first
affinity substance only if there are biomolecules to be detected in
a sample (in a specimen). Next, magnetic beads bind to biomolecules
via the second affinity substance and thus the magnetic beads are
fixed to the surface of the protective film.
[0090] Examples of the biomolecules to be detected include nucleic
acids (that may be naturally derived or chemically synthesized)
such as DNA, mRNA, miRNA, siRNA, and artificial nucleic acids (for
example, locked nucleic acid (LNA), bridged nucleic acid (BNA));
peptides such as ligands, cytokines, and hormones; proteins such as
receptors, enzymes, antigens, and antibodies; cells, viruses,
bacteria, and fungi.
[0091] Examples of the sample containing biomolecules to be
detected include blood, serum, plasma, urine, buffy coat, saliva,
semen, thoracic exudates, cerebrospinal fluids, tears, sputum,
mucosa, lymph fluids, abdominal fluids, pleural effusion, amniotic
fluids, bladder irrigation fluids, bronchoalveolar lavage fluids,
cell extraction liquids, and cell culture supernatants.
[0092] In addition, the biomolecules to be detected may be
biomolecules to be detected with which other biomolecules are
complexed or biomolecules to be detected which are converted into
other biomolecules. For example, a complex obtained by hybridizing
DNA having biotin at the end with RNA (hereinafter referred to as
an "RNA-DNA-biotin complex") may be exemplified. When biotin is
added to RNA by complexation, it can be specifically bound to
streptavidin. Therefore, for example, when RNA or DNA that is
hybridizable with a nucleic acid part in which RNA or DNA contained
in the RNA-DNA-biotin complex is not hybridized is used as the
first affinity substance, they are captured on the biosensor of the
present embodiment. In addition, when streptavidin is used as the
second affinity substance, it is possible to specifically detect
the RNA-DNA-biotin complex.
[0093] For the first affinity substance and the second affinity
substance which specifically bind to biomolecules, when
biomolecules to be detected are nucleic acids, nucleic acids
complementary to the nucleic acids may be exemplified. When
biomolecules to be detected are antigens, antibodies having
affinity for antigens may be exemplified. When biomolecules to be
detected are primary antibodies, antigens having affinity for
primary antibodies and secondary antibodies may be exemplified.
When biomolecules to be detected are cells, viruses, bacteria,
fungi, or the like, antibodies that recognize antigens presented on
surfaces thereof may be exemplified.
[0094] When biomolecules to be detected are miRNA present in blood,
for example, a first nucleic acid complementary to 10 bases at the
5' end of miRNA may be exemplified as the first affinity substance
and a second nucleic acid complementary to 10 bases at the 3' end
of miRNA may be exemplified as the second affinity substance.
[0095] When biomolecules to be detected are antigen proteins
present in blood, for example, first antibodies that recognize the
antigen proteins may be exemplified as the first affinity
substance, and second antibodies that recognize the antigen
proteins and have a different epitope from the first antibodies may
be exemplified as the second affinity substance.
[0096] When the first affinity substance is an antibody, the
antibody can be prepared, for example, by immunizing a rodent
animal such as a mouse with a labeled peptide as an antigen. In
addition, for example, this can be prepared by phage library
screening. The antibody may be an antibody fragment and Fv, Fab,
scFv, and the like may be exemplified as the antibody fragment.
[0097] While an example in which magnetic beads are bound to
biomolecules fixed to the surface of the protective film has been
exemplified in the above description, the present embodiment is not
limited thereto. Biomolecules to be detected may be bound to
magnetic beads in advance, and they may be brought into contact
with the surface of the protective film as a sample.
[0098] As a method of fixing magnetic beads to the surface of the
protective film covering the magnetoresistance effect element, any
technology that has been developed in the past or will be developed
in the future can be applied and any method may be used as long as
it is possible to indirectly detect the presence of biomolecules to
be detected by measuring magnetic beads.
<<Method of Fixing Affinity Substance to Protective
Film>>
[0099] As a method of fixing an affinity substance to a surface of
a protective film, for example, when a constituent material of the
protective film disposed on the top part is a noble metal, a thiol
group, an isothiocyanate group or a disulfide group derived from
the affinity substance or introduced into the affinity substance
forms a thiolate bond with the surface of the noble metal, and the
affinity substance can be fixed.
[0100] In addition, when a constituent material of the protective
film disposed on the top part is an oxide, it is possible to fix
the affinity substance via a silane coupling agent or phosphonic
acid derivatives having a functional group that can bind to the
affinity sub stance.
[0101] The functional group is not particularly limited as long as
it is a group that can be covalently bonded or non-covalently
bonded to the affinity substance. For example, a chemically active
(that is, activated so that the reactivity with the first affinity
substance becomes higher) group, a receptor group, and a ligand
group may be exemplified. The affinity substance may be modified so
that a covalent bond or non-covalent bond with a functional group
can be made.
[0102] As a specific example, an activated carboxyl-derived group,
a carboxyl group, an aldehyde group, an epoxy group, a vinyl
sulfone group, a biotinyl group, a thiol group, an amino group, an
isocyanate group, an isothiocyanate group, a hydroxyl group, an
acrylate group, a maleimide group, a hydrazide group, an aminooxy
group, an azide group, an amide group, a sulfonate group, avidin,
streptavidin, and a metal chelate may be exemplified, but the
present disclosure is not limited thereto. Among these, generally,
since many of the affinity substances have an amino group, in
consideration of the reactivity with the amino group, an aldehyde
group, an activated carboxyl-derived group, an epoxy group, and a
vinylsulfone group are preferable and a biotinyl group having a
high coupling constant is preferable. In particular, when the first
affinity substance has an amino group and is bonded via the amino
group, an activated carboxyl-derived group is preferable in
consideration of the balance between the reactivity with the amino
group and the storage stability. On the other hand, when first
affinity substance has an aldehyde group and is bonded via the
aldehyde group, since the reactivity is high, an aminooxy group or
a hydrazide group is preferable.
[0103] As the phosphonic acid derivatives, for example,
alkylphosphonic acid, alkenylphosphonic acid, and phenylphosphonic
acid may be exemplified. As the phosphonic acid derivatives, more
specifically, vinylphosphonic acid
(CH.sub.2.dbd.CH--PO.sub.3H.sub.2), propene-1-phosphonic acid
(CH.sub.3--CH.dbd.CH--PO.sub.3H.sub.2), and propene-2-phosphonic
acid (CH.sub.2.dbd.CH(CH.sub.3)--PO.sub.3H.sub.2) may be
exemplified and those into which a functional group is introduced
may be used. As phosphonic acid derivatives having a functional
group, for example, 2,5-dicarboxyphenylphosphonic acid,
3,5-dicarboxyphenylphosphonic acid, and
2,5-bisphosphonoterephthalic acid may be exemplified.
[0104] As a silane coupling agent having a functional group, for
example, trimethoxysilylbenzoic acid,
.gamma.-methacryloxypropyltrimethoxysilane, vinyltriacetoxysilane,
vinyltrimethoxysilane, .gamma.-isocyanatopropyltriethoxysilane,
.gamma.-glycidoxypropyltrimethoxysilane,
.beta.-(3,4-epoxycyclohexyl)ethyltrimethoxysilane,
tris-(3-trimethoxysilylpropyl)isocyanurate,
methacryloxypropyldimethylmethoxysilane,
methacryloxypropyldimethylethoxysilane,
methacryloxypropylmethyldimethoxysilane,
methacryloxypropylmethyldiethoxysilane,
methacryloxypropyltrimethoxysilane,
methacryloxypropyltriethoxysilane,
3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane,
3-mercaptopropylmethyldimethoxysilane,
3-mercaptopropylmethyldiethoxysilane,
3-mercaptopropyldimethylmethoxysilane,
3-mercaptopropyldimethylethoxysilane, and
mercaptoethyltriethoxysilane may be exemplified.
[0105] A more specific method of fixing the affinity substance to
the surface of the protective film can be determined by those
skilled in the art using known methods according to a type of the
affinity substance. For example, a method in which a solution
containing an affinity substance is brought into contact with a
protective film together with a silane coupling agent or phosphonic
acid derivatives having a functional group that covalently bonds
with the affinity substance may be exemplified.
[0106] For example, when a material constituting the protective
film is an oxide and an affinity substance having an amino group is
fixed via a silane coupling agent having a carboxyl group, if the
surface of the protective film that is in contact with a solution
in which a first affinity substance and a silane coupling agent are
mixed into a general buffer solution with a pH of 7.0 or more and
10.0 or less is incubated for a predetermined time, the amino group
of the affinity substance and the carboxyl group of the silane
coupling agent react to form an amide bond, and additionally the
silane coupling agent and the surface of the protective film react
to form an ether bond, and thus the affinity substance can be fixed
to the outer surface of the protective film. Examples of the buffer
solution include a phosphate buffer solution, and a tris buffer
solution.
[0107] Adsorption Prevention Film
[0108] The adsorption prevention film is not particularly limited
as long as it can prevent biomolecules or magnetic beads from being
adsorbed between thin lines of the magnetoresistance effect element
having a meander structure. Examples of the material of the
adsorption prevention film include oxides such as alumina, silica,
titanium oxide, zirconium oxide, indium oxide, tartaric oxide, zinc
oxide, gallium oxide, and tin oxide; and inorganic substances of
noble metals such as gold, silver, platinum, rhodium, ruthenium,
and palladium.
[0109] In addition, when a material constituting the protective
film is a noble metal, a material constituting the adsorption
prevention film is preferably an oxide. On the other hand, when a
material constituting the protective film is an oxide, a material
constituting the adsorption prevention film is preferably a noble
metal.
[0110] When materials constituting the protective film and the
adsorption prevention film are different from each other and
additionally combined as above, the affinity substance is
selectively fixed only to the protective film. In addition, the
adsorption prevention film is substantially free of the affinity
substance, and nonspecific adsorption of biomolecules or magnetic
beads on the adsorption prevention film is reduced. Thus, a
measurement error due to magnetic beads present both on thin lines
and between thin lines of the magnetoresistance effect element
having a meander structure is avoided, and it is possible to detect
biomolecules in a sample with high accuracy.
[0111] The adsorption prevention film may be composed of one layer
(single layer) or a plurality of layers such as two or more layers.
In addition, when the adsorption prevention film is composed of a
plurality of layers, the plurality of layers may be the same as or
different from each other, and combinations of the plurality of
layers are not particularly limited.
[0112] The thickness of the adsorption prevention film is
preferably 1 nm or more and 1000 nm or less, more preferably 1 nm
or more and 100 nm or less, and particularly preferably 1 nm or
more and 15 nm or less.
[0113] Here, "the thickness of the adsorption prevention film"
refers to the thickness of the entire adsorption prevention film.
For example, the thickness of the adsorption prevention film
composed of a plurality of layers refers to the total thickness of
all layers constituting the adsorption prevention film.
[0114] In addition, the adsorption prevention film preferably
contains a substance (nonspecific adsorption inhibiting substance)
that reduces nonspecific adsorption of biomolecules on the outer
surface. When the adsorption prevention film contains a nonspecific
adsorption inhibiting substance, it is possible to more effectively
reduce nonspecific adsorption of biomolecules or magnetic
beads.
[0115] The nonspecific adsorption inhibiting substance may be any
monomer or polymer as long as it has a fixing group on the
adsorption prevention film at an end and has a biocompatible group
at the other end or in a compound.
[0116] When a material constituting the adsorption prevention film
is a noble metal, examples of the fixing group include a thiol
group, an isothiocyanate group, and a disulfide group. On the other
hand, when a material constituting the adsorption prevention film
is an oxide, examples of the fixing group include an alkoxysilane
group and a phosphonic acid group.
[0117] The biocompatible group has an excellent nonspecific
adsorption inhibiting effect. As the biocompatible group,
specifically, a phosphorylcholine group, (poly)alkylene glycol
residues, a sulfoalkyl amino group, and the like may be
exemplified. As the nonspecific adsorption inhibiting substance in
the present embodiment, for example, a polymer compound having a
biocompatible group that is produced by polymerizing monomers
having such a biocompatible group may be produced and used.
[0118] When the nonspecific adsorption inhibiting substance is a
monomer, the nonspecific adsorption inhibiting substance is
subjected to molecular self assembly (MSA) and forms a
monomolecular film.
[0119] In this specification, "molecular self assembly" means that
tissues or structures are naturally formed by molecules themselves
without being controlled by external factors.
[0120] In the nonspecific adsorption inhibiting substance in the
present embodiment, a weak intermolecular bond such as Van der
Waals coupling is used, nonspecific adsorption inhibiting
substances are aligned and bonded to form one monomolecular film
(self-assembled monolayers (SAMs)).
[0121] Examples of the monomer having a phosphorylcholine group
include (meth)acryloyloxyalkylphosphorylcholines such as
2-methacryloyloxyethyl phosphorylcholine and
6-methacryloyloxyhexylphosphorylcholine;
(meth)acryloyloxyalkoxyalkylphosphorylcholines such as
2-methacryloyloxyethoxyethyl phosphorylcholine and
10-methacryloyloxyethoxy nonyl phosphorylcholine; and alkenyl
phosphorylcholines such as allyl phosphorylcholine,
butenylphosphorylcholine, hexenyl phosphorylcholine, octenyl
phosphorylcholine, and decenyl phosphorylcholine. As the
nonspecific adsorption inhibiting substance of the present
embodiment, those in which the fixing group is introduced into an
end opposite to the phosphocholine group may be used.
[0122] In this specification, "alkylene glycol residue" refers to
an alkyleneoxy group (--R--O--, here, R denotes an alkylene group)
which remains after hydroxyl groups at one end or both ends of an
alkylene glycol (HO--R--OH, here, R denotes an alkylene group)
undergo a condensation reaction with another compound. For example,
in the case of methylene glycol (HO--CH.sub.2--OH), the alkylene
glycol residue is a methyleneoxy group (--CH.sub.2--O--), and in
the case of ethylene glycol (HO--CH.sub.2CH.sub.2--OH), the
alkylene glycol residue is an ethyleneoxy group
(--CH.sub.2CH.sub.2--O--). In addition, "polyalkylene glycol
residue" refers to a structure in which a plurality of alkyleneoxy
groups are repeated.
[0123] Examples of the monomer having an alkylene glycol residue
include methoxy polyethylene glycol (meth)acrylate,
ethoxypolyethylene glycol (meth)acrylate, 2-hydroxyethyl
(meth)acrylate and monosubstituted esters of its hydroxyl group,
2-hydroxypropyl (meth)acrylate and monosubstituted esters of its
hydroxyl group, 2-hydroxybutyl (meth)acrylate and monosubstituted
esters of its hydroxyl group, glycerol mono (meth)acrylate,
(meth)acrylate having polypropylene glycol as side chain,
2-methoxyethyl (meth)acrylate, 2-ethoxyethyl (meth)acrylate,
methoxydiethylene glycol (meth)acrylate, ethoxydiethyleneglycol
(meth)acrylate, and ethoxypolyethylene glycol (meth)acrylate. In
addition, the average number of repetitions of the alkylene glycol
residue is preferably 5 or more and 90 or less.
[0124] When the average number of repetitions of the alkylene
glycol residue is within the above range, excellent operability
(handling) during synthesis is obtained.
[0125] As the nonspecific adsorption inhibiting substance of the
present embodiment, those in which the fixing group is introduced
into any end of the monomer having the alkylene glycol residue may
be used.
[0126] Examples of the monomer having a sulfoalkyl amino group
include N-methyl-N-(3-sulfopropyl)acrylamide,
3-(N,N-dimethylmyristylammonio)propanesulfonic acid
(3-(N,N-dimethylmyristylammonio)propanesulfonate, and SB3-14,
myristyl sulfobetaine). As the nonspecific adsorption inhibiting
substance of the present embodiment, those in which the fixing
group is introduced into an end opposite to such a sulfo group may
be used.
[0127] As a method of introducing the fixing group into a monomer
having the biocompatible group, a known method may be used
according to a fixing type to be introduced. For example, when the
fixing group is a thiol group, within the monomer having the
biocompatible group, at least one hydrogen bonded to carbon into
which a fixing group is introduced is substituted with a halogen
atom (for example, chlorine, bromine, iodine, etc.), and hydrogen
sulfide is then reacted in the presence of alkali, and thus a thiol
group can be introduced.
[0128] Alternatively, the biocompatible group may be introduced
into the silane coupling agent or phosphocholine derivatives
exemplified in the above <<Method of fixing affinity
substance to protective film>> using a known method.
[0129] As the nonspecific adsorption inhibiting substance of the
present embodiment, more specifically, for example,
3-[(11-mercaptoundecyl)-N,N-dimethylammonio] propanesulfonate which
is a sulfobetaine type alkane thiol may be exemplified.
<<Method of Fixing Nonspecific Adsorption Inhibiting
Substance to Adsorption Prevention Film>>
[0130] As a method of fixing the nonspecific adsorption inhibiting
substance to the surface of the adsorption prevention film, for
example, when a constituent material of the adsorption prevention
film is a noble metal, a nonspecific adsorption inhibiting
substance having a thiol group, an isothiocyanate group or a
disulfide group as a fixing group may be used, the fixing group and
the surface of the noble metal form a thiolate bond, and the
nonspecific adsorption inhibiting substance can be fixed.
[0131] In addition, when a constituent material of the adsorption
prevention film is an oxide, a nonspecific adsorption inhibiting
substance having an alkoxysilane group or a phosphonic acid group
as a fixing group may be used and the fixing group and the surface
of the oxide form an ether bond, and the nonspecific adsorption
inhibiting substance can be fixed.
[0132] A more specific method of fixing the nonspecific adsorption
inhibiting substance to the surface of the adsorption prevention
film can be determined by those skilled in the art by a known
method according to a constituent material of the adsorption
prevention film. For example, a method in which a solution
containing a nonspecific adsorption inhibiting substance is brought
into contact with an adsorption prevention film may be
exemplified.
[0133] For example, when a material constituting the protective
film is an oxide and a nonspecific adsorption inhibiting substance
having an alkoxysilane group is fixed, if the surface of the
adsorption prevention film that is in contact with a solution in
which a nonspecific adsorption inhibiting substance is mixed into a
general buffer solution with a pH of 7.0 or more and 10.0 or less
is incubated for a predetermined time, the nonspecific adsorption
inhibiting substance can be fixed to the surface of the adsorption
prevention film via an ether bond. Examples of the buffer solution
include a phosphate buffer solution, and a tris buffer
solution.
[0134] Other Components
[0135] Electrode Terminal
[0136] The electrode terminal may be disposed on the same plane as
the magnetoresistance effect element or disposed on a plane
different from the magnetoresistance effect element. The electrode
terminal is connected to the magnetoresistance effect element
through contact therewith and can output a change in resistance of
the magnetoresistance effect element as an output to the outside.
In addition, when the electrode terminal is disposed on a plane
different from the magnetoresistance effect element, it may be
disposed directly above the magnetoresistance effect element, and
connected to the magnetoresistance element through contact
therewith (refer to FIG. 1), or it may be disposed directly below
the magnetoresistance effect element and connected to the
magnetoresistance element through contact therewith. For example, a
conductive metal such as Au, Al, Ag, or Cu or an alloy thereof is
preferably used as a material of the electrode terminal.
[0137] Insulating Layer
[0138] When the substrate is made of a conductive material, the
insulating layer is formed on the main surface of the substrate and
an electrical short circuit via the substrate can be prevented. For
example, an inorganic substance such as alumina, aluminum nitride,
silicon oxide, or silicon nitride or an organic substance such as a
polyimide is preferably used as a material of the insulating
layer.
[0139] Applied Magnetic Field and Detection Magnetic Field
[0140] When magnetic beads accumulate on the protective film via
biomolecules and a magnetic field (applied magnetic field) is
horizontally applied as shown in FIG. 2, a detection magnetic field
(stray magnetic field) is input to the magnetoresistance effect
element. A direction of the applied magnetic field is preferably a
direction crossing the main surface of the magnetoresistance effect
element. The applied magnetic field is not particularly limited,
and is preferably 0.1 m tesla or more and 100 m tesla or less and
more preferably 1 m tesla or more and 10 m tesla or less.
[0141] The detection magnetic field (stray magnetic field) is
influenced by a proportion of magnetic beads occupying the main
surface of the magnetoresistance effect element via the protective
film. As the number of magnetic beads accumulated on the protective
film increases, a detected resistance value changes. The number of
magnetic beads accumulated on the protective film and the detected
resistance value via the stray magnetic field are linearly
correlated.
[0142] Then, based on the titer (for example, the number of
biomolecules that the second affinity substance captures) of the
second affinity substance included in the magnetic beads, it is
possible to calculate the number of biomolecules accumulated on the
protective film.
[0143] That is, according to the biosensor of the present
embodiment, the number of biomolecules contained in a sample can be
calculated. In this manner, in the biosensor of the present
embodiment, it is possible to secure quantitation of biomolecules
in a sample with high accuracy.
[0144] In addition, the biosensor of the present embodiment has
high sensitivity, and can perform detection at a level of tens of
nanotesla. Specifically, it is possible to detect an increase or
decrease of 10 with respect to 1500 magnetic beads. That is, a
change of about 0.5% can be detected.
[0145] In addition, since the biosensor of the present embodiment
uses magnetic beads, it has higher sensitivity and a longer
lifespan compared to fluorescence. Therefore, it is much better
than a detection method such as ELISA.
[0146] Method of Producing Biosensor
[0147] The biosensor of the present embodiment can be produced
using a known method according to sequential lamination so that it
has a corresponding positional relationship among the above
components.
[0148] In addition, a method of fixing an affinity substance to a
protective film is the same as the <<Method of fixing
affinity substance to protective film>> described above.
[0149] In addition, a method of fixing a nonspecific adsorption
inhibiting substance to an adsorption prevention film is the same
as the <<Method of fixing nonspecific adsorption inhibiting
substance to adsorption prevention film>> described
above.
[0150] Method of Using Biosensor
[0151] Method of Detecting Biomolecules
[0152] The biosensor of the present embodiment can be used for, for
example, a method of detecting biomolecules to be described
below.
[0153] First, a sample containing biomolecules is brought into
contact with a protective film, and the biomolecules accumulate on
the protective film via the first affinity substance (Process 1).
Next, magnetic beads are brought into contact with the protective
film and accumulate on the protective film via the biomolecules
(Process 2). Next, a magnetic field is applied in a direction
crossing the main surface of the magnetoresistance effect element,
a detection magnetic field is input to the magnetoresistance effect
element, and a resistance value is detected (Process 3).
[0154] The processes will be described in detail.
[Process 1]
[0155] Process 1 is a process in which a sample containing
biomolecules is brought into contact with a protective film and the
biomolecule accumulate on the protective film via a first affinity
substance. In consideration of convenience and the like, the
biosensor is preferably used in a microfluidic device. In Process
1, first, a sample containing biomolecules flow through a micro
flow path. The sample not particularly limited as long as it
contains biomolecules to be detected. As a sample, for example,
when the method of detecting biomolecules of the present embodiment
is used for diagnosis of a disease, a sample derived from a subject
such as a person in whom onset of a disease was confirmed or a
person in whom onset of a disease was suspected, or a sample
derived from a subject such as a patient being treated for a
disease may be exemplified. As the sample, more specifically, the
same as those exemplified in ".smallcircle. Protective film" above
may be used.
[0156] For example, when peptides and proteins such as antigens and
receptors present on the surface of blood circulating tumor cells
are to be detected, a sample may be caused to directly flow through
the micro flow path. For example, it has been reported that miRNA
is involved in onset and progress of cancer, cardiovascular
diseases, neurodegenerative diseases, mental illness, chronic
inflammatory diseases and the like. When nucleic acids such as
genomic DNA, cDNA, Total RNA, mRNA, and rRNA including miRNA are to
be detected, a nucleic acid is preferably extracted from the
biological sample. The extraction method is appropriately selected
from conventional methods according to a type of nucleic acid.
[0157] Biomolecules in a sample which flows through a micro flow
path are captured by the first affinity substance on the protective
film and accumulate on the protective film. As the first affinity
substance, nucleic acids, antibodies, and the like may be
exemplified as described above. The biomolecules form a complex
with the first affinity substance on the protective film according
to hybridization, an antigen and antibody reaction, and the
like.
[0158] After the first affinity substance-biomolecule complex is
formed on the protective film, the protective film is preferably
washed using a buffer solution or the like. According to washing,
impurities that are nonspecifically bound to the protective film
can be removed and detection accuracy of the biomolecules can be
improved. Examples of the buffer solution include a phosphate
buffer solution, and a tris buffer solution.
[Process 2]
[0159] Process 2 is a process in which magnetic beads are brought
into contact with a protective film and accumulate on the
protective film via the biomolecules. As described above, the
magnetic beads contain a second affinity substance that captures
biomolecules. For example, when magnetic beads flow through a micro
flow path and come in contact with the protective film, they bind
to biomolecules in the first affinity substance-biomolecule complex
formed on the protective film via the second affinity substance. In
Process 2, a first affinity substance-biomolecule-second affinity
substance complex is formed on the protective film. That is, the
magnetic beads containing the second affinity substance accumulate
on the protective film.
[0160] After the first affinity substance-biomolecule-second
affinity substance complex is formed on the protective film, the
protective film is preferably washed with a buffer solution or the
like as in Process 1. According to washing, the magnetic beads that
are nonspecifically bound to the protective film can be removed,
and detection accuracy of the biomolecule can be improved. As the
buffer solution, the same ones exemplified in [Process 1] may be
exemplified.
[Process 3]
[0161] Process 3 is a process in which a magnetic field is applied
in a direction crossing the main surface of the magnetoresistance
effect element, a detection magnetic field is input to the
magnetoresistance effect element, and a resistance value is
detected.
[0162] The detection magnetic field (stray magnetic field) is
influenced by a proportion of magnetic beads occupying the main
surface of the magnetoresistance effect element via the protective
film. As the number of magnetic beads accumulated on the protective
film increases, a detected resistance value increases.
[0163] According to Process 3, it is possible to quantify
accurately the number of magnetic beads accumulated on the
protective film. Then, based on the titer (for example, the number
of biomolecules that the second affinity substance captures) of the
second affinity substance included in the magnetic beads, it is
possible to calculate the number of all biomolecules accumulated on
the protective film. That is, according to the detection method of
the present embodiment, it is possible to calculate the number of
biomolecules contained in a sample. Therefore, when there is a
positive correlation between the number of biomolecules in a sample
and a disease state, if the number of biomolecules in the sample is
successively calculated, it is possible to perform follow-up
observation of the disease state.
[0164] As described above, in the detection method of the present
embodiment, it is possible to secure quantitation of biomolecules
in a sample.
[0165] As another use example of the biosensor of the present
embodiment, a method of detecting biomolecules to be described
below may be used.
[0166] First, a sample containing biomolecules and magnetic beads
are mixed tighter and the biomolecules are captured by the magnetic
beads via the second affinity substance (Process 4). Next, magnetic
beads that have captured biomolecules are brought into contact with
a protective film and the magnetic beads accumulate on the
protective film via the biomolecules (Process 5). Next, a magnetic
field is applied in a direction crossing the magnetoresistance
effect element, a detection magnetic field is input to the
magnetoresistance effect element, and a resistance value is
detected (Process 3).
[0167] Since this method is the same as the method of detecting
biomolecules including the above [Process 1] to [Process 3] except
that, when a first affinity substance-biomolecule-second affinity
substance complex is formed, a biomolecule-second affinity
substance complex is formed in advance, description thereof will be
omitted.
[0168] Biochip
[0169] The biosensor of the present embodiment can be applied to a
biochip.
[0170] When a plurality of biosensors with different first affinity
substances on the protective film are provided, the biochip of the
present embodiment can comprehensively analyze properties of a
sample.
[0171] As the biochip, for example, a biochip for cancer diagnosis,
a biochip for carcinoma diagnosis, and a biochip for detecting
influenza virus may be exemplified.
[0172] Biochip for Cancer Diagnosis
[0173] As the first affinity substance provided on the protective
film, a nucleic acid complementary to a nucleic acid derived from a
cancer gene or a cancer inhibiting gene may be exemplified. When
there is a mutation specific to a cancer patient in the cancer gene
or the cancer inhibiting gene, a nucleic acid complementary to the
nucleic acid containing the mutation is preferable.
[0174] As the cancer gene, a gene group that encodes a growth
factor such as sis; a gene group that encodes a receptor type
tyrosine kinase such as erbB, fms, and ret; a gene group that
encodes a non-receptor type tyrosine kinase such as fes; a gene
group that encodes a GTP/GDP binding protein such as ras; a gene
group that encodes a serine/threonine kinase such as src, mos, and
raf; a gene group that encodes a nuclear protein such as myc, myb,
fos, jun, and erbA; a gene group that encodes a signal transducing
adapter molecule such as crk; and a fusion gene such as Bcr-Abl may
be exemplified.
[0175] In addition, as the cancer gene, a Ras-MAP kinase
pathway-linked gene such as Shc, Grb2, Sos, MEK, Rho, and Rac
genes; a phospholipase C gamma-protein kinase C pathway-linked gene
such as PLCy and PKC; a PI3K-Akt pathway-linked gene such as PI3K,
Akt, and Bad; a JAK-STAT pathway-linked gene such as JAK and STAT;
and a GAP-related pathway-linked gene such as GAP, p180, and p62
may be exemplified.
[0176] Examples of the cancer inhibiting gene include RB, p53, WT1,
NF1, APC, VHL, NF2, p16, p19, BRCA1, BRCA2, PTEN, and E cadherin
gene.
[0177] In addition, as the first affinity substance, a substance
that captures a protein which is a genetic product of the above
genes, for example, an antibody (including an antibody fragment),
an aptamer, a ligand, and a receptor may be used.
[0178] Biochip for Diagnosis for Specific Type of Cancer
[0179] In the biochip of the present embodiment, a first affinity
substance provided on a protective film may be a nucleic acid
complementary to a plurality of nucleic acids that are extracted
from one type of cancer. That is, the biochip of the present
embodiment may be a biochip for diagnosis of a specific type of
cancer.
[0180] The target cancer is not particularly limited, and, for
example, breast cancer (for example, invasive ductal carcinoma,
noninvasive ductal carcinoma, Inflammatory breast cancer, etc.),
prostate cancer (for example, hormone-dependent prostate cancer,
hormone-independent prostate cancer, etc.), pancreatic cancer (for
example, pancreatic duct cancer, etc.), stomach cancer (for
example, papillary adenocarcinoma, mucinous adenocarcinoma,
adenosquamous carcinoma, etc.), lung cancer (for example, non small
cell lung cancer, small cell lung cancer, malignant mesothelioma,
etc.), colon cancer (for example, familial colorectal cancer,
hereditary nonpolyposis colorectal cancer, gastrointestinal stromal
tumor, etc.), rectal cancer (for example, gastrointestinal stromal
tumor, etc.), small intestine cancer (for example, non-Hodgkin's
lymphoma, gastrointestinal stromal tumor, etc.), small intestine
cancer (for example, non-Hodgkin's lymphoma, gastrointestinal
stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue
cancer, pharyngeal cancer (for example, nasopharyngeal cancer,
oropharyngeal cancer, hypopharyngeal cancer, etc.), head and neck
cancer, salivary gland cancer, brain tumor (for example, pineal
gland stellate cell tumor, pilocytic astrocytoma, diffuse
astrocytoma, anaplestic astrocytoma, etc.), schwannoma, liver
cancer (for example, primary liver cancer, extrahepatic bile duct
cancer, etc.), kidney cancer (for example, renal cell carcinoma,
transitional epithelial carcinoma of the renal pelvis and ureter,
etc.), gall bladder cancer, bile duct cancer, pancreatic cancer,
liver cancer, endometrial cancer, cervical cancer, ovarian cancer
(for example, epithelial ovarian cancer, extragonadal germ cell
tumor, ovarian germ cell tumor, ovarian low grade tumor, etc.),
bladder cancer, urethral cancer, skin cancer (for example,
intraocular (eye) melanoma, Merkel cell cancer, etc.), hemangioma,
malignant lymphoma (for example, reticulosarcoma, lymphosarcoma,
Hodgkin's disease, etc.), melanoma (malignant melanoma), thyroid
cancer (for example, medullary thyroid cancer, etc.), parathyroid
cancer, nasal cancer, paranasal sinus cancer, bone tumor (for
example, osteosarcoma, Ewing's tumor, uterine sarcoma, soft tissue
sarcoma, etc.), metastatic medulloblastoma, angiofibroma,
dermatofibrosarcoma protuberans, retinosarcoma, penis cancer,
testicular tumor, pediatric solid tumor (for example, Wilms tumor,
pediatric renal tumor, etc.), Kaposi's sarcoma, Kaposi's sarcoma
caused by AIDS, maxillary sinus neoplasm, fibrous histiocytoma,
leiomyosarcoma, rhabdomyosarcoma, chronic myeloproliferative
disease, and leukemia (for example, acute myelogenous leukemia,
acute lymphoblastic leukemia, etc.) may be exemplified and the
target cancer is not limited thereto.
[0181] It has been reported that there is a specific gene
expression/mutation pattern according to a cancer type including
the cancer gene and cancer inhibiting gene described above.
Therefore, when a biochip of the present embodiment is prepared on
the basis of a gene expression profile for each cancer type and the
like, it is possible to increase the accuracy of diagnosis.
[0182] In addition, when the biochip of the present embodiment is
used, it is possible to predict the susceptibility/resistance of an
anti-cancer agent. For example, it has been reported that, in the
case of gefinitib which is an EGFR inhibitor, when EGFR in a test
sample has an L858R mutation or G719X mutation, the mutation
exhibits gefinitib susceptibility.
[0183] On the other hand, it has been reported that, when EGFR in a
test sample has a T790M mutation and/or D761Y mutation, the
mutation exhibits gefitinib resistance. In addition, it has been
reported that these mutations exhibiting gefitinib resistance
detected at a higher frequency as a stage of disease progresses. In
the biochip of the present embodiment, since it is possible to
easily quantify an EGRF gene exhibiting a resistance mutation,
according to the biochip of the present embodiment, it is possible
to check a degree of progress of cancer.
[0184] Influenza Virus Detection Biochip
[0185] In addition, in the biochip of the present embodiment, as
the first affinity substance provided on the protective film, a
nucleic acid complementary to a nucleic acid derived from influenza
virus or a carbohydrate chain to which influenza virus specifically
binds may be exemplified. That is, the biochip of the present
embodiment may be a biochip for detecting influenza virus.
[0186] As the biochip of the present embodiment, for example, in A
type, B type, and C type genomes, a nucleic acid that recognizes a
certain mutation site including a reported mutation and is fixed to
a protective film may be exemplified. In addition, as the first
affinity substance, an antibody that can specifically recognize A
type, B type and C type viruses may be used. In addition, it is
known that influenza virus binds to a sialic acid residue when a
cell is infected therewith, and since binding modes of a sialic
acid to which virus can bind and a sugar differ according to a type
of virus, as the first affinity substance, a sialic acid-containing
carbohydrate chain to which A type, B type and C type viruses bind
may be used.
[0187] Here, in this specification, "sialic acid" generally refers
to a substance in which an amino group or a hydroxy group of a
nine-carbon sugar neuraminic acid is substituted. For example,
N-acetylneuraminic acid (Neu5Ac) acetylated in position 5 and
N-glycolylneuraminic acid (Neu5Gc) modified with a glycolic acid
may be exemplified.
[0188] According to the biochip of the present embodiment, it is
possible to detect infection of influenza virus at an early
stage.
[0189] In addition, when the biochip of the present embodiment is
used over time, it is possible to perform follow-up observation of
a disease state after viral infection.
INDUSTRIAL APPLICABILITY
[0190] According to the above embodiment, a measurement error due
to magnetic beads present both on thin lines and between thin lines
of the magnetoresistance effect element having a meander structure
is avoided, and it is possible to detect biomolecules in a sample
with high accuracy. In addition, when the biosensor of the present
embodiment is used as a biochip, it is possible to perform
diagnosis easily and rapidly, and it can be applied for, for
example, cancer diagnosis, diagnosis for a specific type of cancer,
diagnosis of a degree to which cancer has progressed, detection of
influenza virus, identification of a type of influenza virus, and
observation of a state of an influenza disease.
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