U.S. patent application number 16/860808 was filed with the patent office on 2020-08-13 for method of manufacturing sensing chip and sensing chip.
This patent application is currently assigned to KONICA MINOLTA, INC.. The applicant listed for this patent is KONICA MINOLTA, INC.. Invention is credited to Hiroshi HIRAYAMA, Tomonori KANEKO, Takatoshi KAYA, Kosuke NAGAE, Yukito NAKAMURA.
Application Number | 20200256796 16/860808 |
Document ID | / |
Family ID | 57144597 |
Filed Date | 2020-08-13 |
United States Patent
Application |
20200256796 |
Kind Code |
A1 |
NAKAMURA; Yukito ; et
al. |
August 13, 2020 |
METHOD OF MANUFACTURING SENSING CHIP AND SENSING CHIP
Abstract
A first frame having a first through-hole is arranged on a
support so that one opening of the first through-hole is closed. A
liquid containing a capturer for capturing a substance to be
detected is fed into the first through-hole, and the capturer is
immobilized on the support exposed in the first through-hole. After
removing the liquid from the support, a second frame having the
second through-hole is arranged on the support in the first
through-hole so that one opening of the second through-hole is
closed.
Inventors: |
NAKAMURA; Yukito;
(Tokorozawa-shi, JP) ; KAYA; Takatoshi; (Tokyo,
JP) ; KANEKO; Tomonori; (Tokyo, JP) ; NAGAE;
Kosuke; (Tokyo, JP) ; HIRAYAMA; Hiroshi;
(Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KONICA MINOLTA, INC. |
Tokyo |
|
JP |
|
|
Assignee: |
KONICA MINOLTA, INC.
Tokyo
JP
|
Family ID: |
57144597 |
Appl. No.: |
16/860808 |
Filed: |
April 28, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15558506 |
Sep 14, 2017 |
10677731 |
|
|
PCT/JP2016/061112 |
Apr 5, 2016 |
|
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16860808 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 21/64 20130101;
G01N 21/6428 20130101; G01N 21/552 20130101; G01N 2021/6482
20130101; G01N 2021/6439 20130101; G01N 21/03 20130101; G01N
2201/0612 20130101; G01N 33/54373 20130101; G01N 21/648 20130101;
G01N 33/543 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64; G01N 33/543 20060101 G01N033/543; G01N 21/03 20060101
G01N021/03 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2015 |
JP |
2015-087588 |
Claims
1. A method of manufacturing a sensing chip for detecting a
substance to be detected in a specimen, the method comprising:
arranging a first frame having a first through-hole on a support so
that one opening of the first through-hole is closed; feeding a
liquid containing a capturer for capturing the substance to be
detected in the first through-hole and immobilizing the capturer on
the support exposed in the first through-hole; and removing the
liquid from the support, and thereafter arranging a second frame
having a second through-hole on the support in the first
through-hole so that one opening of the second through-hole is
closed.
2. The method of manufacturing a sensing chip according to claim 1,
wherein the first frame has a light blocking property.
3. The method of manufacturing a sensing chip according to claim 1,
wherein the first frame has a tapered portion where, as the first
frame is away from the support, a cross-sectional area of the first
through-hole increases in a direction perpendicular to a height
direction of the first through-hole.
4. The method of manufacturing a sensing chip according to claim 1,
wherein the second frame has a tapered portion where, as the second
frame is away from the support, a cross-sectional area of the
second through-hole increases in a direction perpendicular to a
height direction of the second through-hole.
5. The method of manufacturing a sensing chip according to claim 1,
wherein a height of the second through-hole is larger than a height
of the first through-hole.
6. The method of manufacturing a sensing chip according to claim 4,
wherein an inner wall surface of the tapered portion of the second
frame is inclined so that light emitted from a center of a surface
of the support exposed in the second through-hole and emitted at an
emitting angle of 20.degree. or less is not blocked.
7. The method of manufacturing a sensing chip according to claim 1,
further comprising performing a blocking treatment on an inner wall
surface of the second through-hole.
8. The method of manufacturing a sensing chip according to claim 1,
wherein at least one of the support, the first frame, and the
second frame has a reagent holder for accommodating a reagent.
9. The method of manufacturing a sensing chip according to claim 1,
wherein at least one of the first frame and the second frame is
made of a resin film.
10. The method of manufacturing a sensing chip according to claim
1, wherein the support is a prism made of a dielectric material and
having a metal film formed on one surface thereof, and the first
frame and the second frame are arranged on the one surface.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional application of U.S.
application Ser. No. 15/558, 506, filed Sep. 14, 2017, which is a
U.S. National Phase application of International Application No.
PCT/JP2016/061112, filed Apr. 5, 2016, which is based upon and
claims the benefit of priority from prior Japanese Patent
Application No. 2015-087588, filed Apr. 22, 2015. The entire
contents of all the above identified applications are incorporated
herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to a method of manufacturing a
sensing chip for detecting a substance to be detected and a sensing
chip.
BACKGROUND ART
[0003] When a trace amount of a substance to be detected such as
protein and DNA can be quantitatively detected with high
sensitivity in clinical examinations or the like, it is possible to
rapidly check the condition of the patient and to treat a disease.
For this reason, there is a demand for a detection apparatus
capable of detecting a trace amount of a substance to be detected
quantitatively with high sensitivity.
[0004] As a detection apparatus capable of detecting a substance to
be detected with high sensitivity, there is known a device
utilizing surface plasmon resonance fluorescence spectroscopy
(Surface Plasmon-field enhanced Fluorescence Spectroscopy:
hereinafter, abbreviated as "SPFS") (for example, refer to Patent
Literature 1).
[0005] In the detection apparatus disclosed in Patent Literature 1,
used is a sensing chip (sensor structure) having a sensor member
and a well member immobilized on the sensor member. The sensor
member includes a prism (dielectric member), a metal film formed on
the prism, and a reaction field (ligand immobilization region)
which is a region where a capturer (ligand) formed on the metal
film for capturing a substance to be detected (analyte) is
immobilized. The well member has a through-hole at a position
corresponding to the reaction field. By arranging the well member
on the sensor member, a well for storing a sample solution is
formed. In the detection apparatus, the capturer captures the
substance to be detected in the reaction field in the well, and the
captured substance to be detected is labeled with a fluorescent
substance. In this state, when the metal film is irradiated with
excitation light through the prism at an angle at which surface
plasmon resonance occurs, localized field light can be generated on
the surface of the metal film. By the localized field light, the
fluorescent substance that labels the substance to be detected
captured on the metal film is selectively excited, and fluorescence
light is emitted from the fluorescent substance. By detecting the
fluorescence light, the detection apparatus can detect the presence
or amount of the substance to be detected.
[0006] In the sensing chip, if the substance to be detected adheres
to an inner wall surface of the through-hole of the well member,
the detection efficiency is lowered. In order to prevent the
problem, the sensing chip disclosed in Patent Literature 1 is
manufactured by immobilizing the capturer on the entire surface of
the metal film, and after that, arranging the well member on the
metal film or by immobilizing the capturer on a portion of the
surface of the metal film by using a frame member having a
through-hole, and after that, removing the frame member and newly
arranging the well member on the metal film. By manufacturing the
sensing chip in this manner, it is possible to prevent the capturer
from being immobilized on the inner wall surface of the
through-hole of the well member.
CITATION LIST
Patent Literature
[0007] Patent Literature 1: WO 2012/157403 A
SUMMARY OF INVENTION
Technical Problem
[0008] However, in the method of manufacturing the sensing chip
disclosed in Patent Literature 1, when the capturer is immobilized
on the entire surface of the metal film, an excessive amount of the
capturer is used, which causes an increase in production cost. In
addition, in a case where the capturer is immobilized on a portion
of the surface of the metal film by using the frame member, since a
step of removing the frame member not constituting the sensing chip
is required, the manufacturing process becomes complicated.
[0009] An object of the present invention is to provide a method of
manufacturing a sensing chip capable of preventing immobilization
of a capturer on an inner wall surface of a well without using a
frame member that does not constitute a sensing chip and the
sensing chip manufactured by the method.
Solution to Problem
[0010] To solve the above problem, a method of manufacturing a
sensing chip according to an embodiment of the present invention is
a method of manufacturing a sensing chip used for detecting a
substance to be detected in a specimen, including: a step of
arranging a first frame having a first through-hole on a support so
that one opening of the first through-hole is closed; a step of
feeding a liquid containing a capturer for capturing a substance to
be detected in the first through-hole and immobilizing the capturer
on the support exposed in the first through-hole; and a step of
removing the liquid from the support, and after that, arranging a
second frame having a second through-hole on the support in the
first through-hole so that one opening of the second through-hole
is closed.
[0011] To solve the above problem, a sensing chip according to an
embodiment of the present invention is a sensing chip used for
detecting a substance to be detected in a specimen, including: a
support; a first frame having a first through-hole and arranged on
the support so that one opening of the first through-hole is
closed; and a second frame having a second through-hole and
arranged on the support in the first through-hole so that one
opening of the second through-hole is closed, wherein a capturer
for capturing the substance to be detected in the specimen is
immobilized on at least a portion of an inner wall surface of the
first through-hole of the first frame and on a surface of the
support exposed in the first through-hole.
Advantageous Effects of Invention
[0012] According to a method of manufacturing a sensing chip
according to the present invention, it is possible to easily
manufacture a sensing chip in which a capturer is not immobilized
on an inner wall surface of a well without using a frame member
that does not constitute the sensing chip. By using the sensing
chip obtained by the present invention, it is possible to detect a
substance to be detected with high sensitivity and high accuracy
while suppressing a loss of the substance to be detected due to
adhesion to the inner wall surface of the well.
BRIEF DESCRIPTION OF DRAWINGS
[0013] FIG. 1 is a diagram illustrating a configuration of a
surface plasmon enhanced fluorescence detection apparatus.
[0014] FIGS. 2A and 2B are diagrams illustrating a configuration of
a sensing chip according to an embodiment of the present
invention.
[0015] FIG. 3 is a flowchart illustrating an example of processes
of manufacturing a sensing chip according to an embodiment of the
present invention.
[0016] FIGS. 4A to 4D are schematic diagrams illustrating an
example of processes of manufacturing a sensing chip according to
an embodiment of the present invention.
[0017] FIG. 5 is a flowchart illustrating an example of an
operation procedure of a surface plasmon enhanced fluorescence
detection apparatus.
[0018] FIGS. 6A and 6B are diagrams illustrating a configuration of
a sensing chip according to Modified Example 1.
[0019] FIGS. 7A and 7B are diagrams illustrating a configuration of
a sensing chip according to Modified Example 2.
DESCRIPTION OF EMBODIMENTS
[0020] Hereinafter, embodiments of the present invention will be
described in detail with reference to the drawings. In the
following description, a surface plasmon enhanced fluorescence
detection apparatus (hereinafter, also referred to as an "SPFS
apparatus") for detecting a substance to be detected by using
surface plasmon resonance (SPR) will be described as a
representative example of a detection apparatus.
[0021] FIG. 1 is a diagram illustrating a configuration of an SPFS
apparatus 100. In FIG. 1, hatching is omitted in order to
illustrate paths of light. As illustrated in FIG. 1, the SPFS
apparatus 100 is configured to include a chip holder 110 for
detachably holding a sensing chip 10, a light irradiation unit 120
for irradiating the sensing chip 10 with light, a light receiving
unit 130 for detecting light (plasmon scattered light .beta. or
fluorescence light .gamma.) emitted from the sensing chip 10, and a
control unit (processing unit) 140 for controlling these
components. The SPFS apparatus 100 is used in the state where the
sensing chip 10 is mounted on the chip holder 110. The sensing chip
10 will be described first, and each component of the SPFS
apparatus 100 will be described thereafter.
[0022] (Configuration of Sensing Chip)
[0023] FIGS. 2A and 2B are diagrams illustrating a configuration of
the sensing chip 10 according to the embodiment. FIG. 2A is a plan
diagram of the sensing chip 10, and FIG. 2B is a cross-sectional
diagram taken along line B-B in FIG. 2A. As illustrated in FIGS. 2A
and 2B, the sensing chip 10 is configured to include a prism
(support) 20 having an incidence surface 21, a film formation
surface 22, and a light emission surface 23 and having a metal film
30 formed on the film formation surface 22, a first frame 40
arranged on the metal film 30 and having a first through-hole 41,
and a second frame 50 arranged in the first through-hole 41 on the
metal film 30 and having a second through-hole 51. By arranging the
second frame 50 on the prism 20 so that one opening of the second
through-hole 51 is closed, a well 53 for accommodating a liquid is
formed.
[0024] The prism 20 is made of a dielectric material that is
transparent to excitation light .alpha.. As described above, the
prism 20 has the incidence surface 21, the film formation surface
22, and the light emission surface 23.
[0025] The incidence surface 21 allows the excitation light .alpha.
from the light irradiation unit 120 to be incident into the
interior of the prism 20. The metal film 30 is arranged on the film
formation surface 22. The excitation light .alpha. incident into
the interior of the prism 20 is reflected by the metal film 30.
More specifically, the excitation light .alpha. is reflected at the
interface (the film formation surface 22) between the prism 20 and
the metal film 30. The light emission surface 23 emits the
excitation light .alpha. reflected by the metal film 30 to the
outside of the prism 20.
[0026] The shape of the prism 20 is not particularly limited. In
the embodiment, the shape of the prism 20 is a columnar body having
a trapezoid as a bottom surface. The surface corresponding to one
bottom side of the trapezoid is the film formation surface 22, the
surface corresponding to one leg is the incidence surface 21, and
the surface corresponding to the other leg is the light emission
surface 23. The trapezoid which is the bottom surface is preferably
an isosceles trapezoid. Therefore, the incidence surface 21 and the
light emission surface 23 are symmetrical, and the S-wave component
of the excitation light .alpha. is hard to stay in the prism 20.
The incidence surface 21 is formed so that the excitation light
.alpha. does not return to the light irradiation unit 120. This is
because, in a case where the light source of the excitation light
.alpha. is a laser diode (hereinafter, also referred to as an
"LD"), when the excitation light .alpha. returns to the LD, the
excited state of the LD is disturbed, and thus, the wavelength and
output of the excitation light .alpha. fluctuate. Therefore, in the
scanning range centered on an ideal resonance angle or enhancement
angle, the angle of the incidence surface 21 is set so that the
excitation light .alpha. is not incident perpendicularly on the
incidence surface 21.
[0027] Herein, the "resonance angle" denotes the incidence angle
when the light amount of the reflected light (not illustrated)
emitted from the light emission surface 23 is at a minimum in the
case of scanning the incidence angle of the excitation light
.alpha. to the metal film 30. In addition, the "enhancement angle"
denotes the incidence angle when the light amount of the scattered
light (hereinafter, referred to as "plasmon scattered light")
.beta. having the same wavelength as the excitation light .alpha.
emitted above the sensing chip 10 is at a maximum in the case of
scanning the incidence angle of the excitation light .alpha. to the
metal film 30. For example, each of the angle between the incidence
surface 21 and the film formation surface 22 and the angle between
the film formation surface 22 and the light emission surface 23 is
about 80.degree..
[0028] The prism 20 may have other elements as required. For
example, the prism 20 may have a reagent holder for accommodating a
reagent at a position which does not interfere with the optical
path of the excitation light .alpha..
[0029] Examples of materials for the prism 20 include resin and
glass. Examples of the resin constituting the prism 20 include
polymethylmethacrylate (PMMA), polycarbonate (PC), and a
cycloolefin-based polymer. The prism 20 is preferably a resin
having a refractive index of 1.4 to 1.6 and a small
birefringence.
[0030] The metal film 30 is formed on one surface (film formation
surface 22) of the prism 20. By providing the metal film 30,
interaction (surface plasmon resonance; SPR) occurs between photons
of the excitation light .alpha. incident on the film formation
surface 22 under the total reflection condition and free electrons
in the metal film 30, so that it is possible to generate the
localized field light on the surface of the metal film 30. The
material of the metal film 30 is not particularly limited as long
as the material of the metal film is a metal that generates surface
plasmon resonance. Examples of the material of the metal film 30
include gold, silver, copper, aluminum, and alloys thereof. In the
embodiment, the metal film 30 is a gold thin film. Although the
thickness of the metal film 30 is not particularly limited, the
thickness of the metal film is preferably in the range of 30 to 70
nm.
[0031] In addition, a capturer 60 for capturing the substance to be
detected is immobilized on the surface of the metal film 30 not
facing the prism 20. More specifically, the capturer 60 is
immobilized on at least a portion of the inner wall surface of the
first through-hole 41 of the first frame 40 and the surface of the
metal film 30 exposed in the first through-hole 41 (refer to FIG.
4C described later). In addition, in FIG. 2B, in order to
illustrate the second frame 50, the capturer 60 immobilized on the
inner wall surface of the first through-hole 41 and the capturer 60
immobilized on the portion of the surface of the metal film 30
where the second frame 50 is arranged are omitted. By the capturer
60, it is possible to selectively detect the substance to be
detected. At least a portion of the surface of the metal film 30 on
which the capturer 60 is immobilized is set as a reaction field
where a reaction such as binding (primary reaction) of the capturer
60 and the substance to be detected or fluorescence labeling
(secondary reaction) of the substance to be detected is performed.
In the embodiment, the surface of the metal film 30 exposed in the
second through-hole 51 of the second frame 50 described later is
set as a reaction field. The type of the capturer 60 is not
particularly limited as long as the capturer can capture the
substance to be detected. For example, the capturer 60 is an
antibody or a fragment thereof capable of specifically binding to a
substance to be detected.
[0032] The first frame 40 has the first through-hole 41 and is
arranged on the prism 20 (the metal film 30) so that one opening of
the first through-hole 41 is closed. When manufacturing the sensing
chip 10 according to the embodiment, the first frame 40 defines a
region on the metal film 30 where the capturer 60 is to be
immobilized. The number, shape and size of the first through-holes
41 are not particularly limited and can be appropriately set
according to the use of the sensing chip 10. In the embodiment, the
number of the first through-holes 41 is one, and the shape of the
first through-holes 41 is a cylindrical shape.
[0033] The first frame 40 preferably has a light blocking property.
Therefore, by cutting noise light such as auto-fluorescence light
emitted from the prism 20 or external light, it is possible to
prevent the noise light from reaching the light receiving unit 130
of the SPFS apparatus 100.
[0034] The outer shape of the first frame 40 is not particularly
limited. For example, the outer shape of the first frame 40 in a
plan view is a circular shape, a quadrilateral shape, or the like.
In the embodiment, the outer shape of the first frame 40 in a plan
view is a quadrilateral shape.
[0035] The first frame 40 may have other elements as required. For
example, as illustrated in FIGS. 2A and 2B, the first frame 40 may
have a reagent holder 43 for accommodating reagents. The reagent
holder 43 may be integral with the first frame 40 or may be a
separate body. In the embodiment, the reagent holder 43 is
integrated with the first frame 40.
[0036] Examples of the material of the first frame 40 include resin
and glass. For example, the first frame 40 may be a resin film.
Therefore, the manufacturing becomes easy, the manufacturing cost
can be reduced, and the sensing chip 10 can be miniaturized.
[0037] The second frame 50 has a second through-hole 51 and is
arranged on the prism 20 (the metal film 30) in the first
through-hole 41 so that one opening of the second through-hole 51
is closed. The second frame 50 may be arranged so as to be in
contact with the first frame 40 without a gap or may be arranged to
be separated from the first frame. The second frame 50 defines the
well 53 for accommodating the liquid. In addition, since the region
(reaction field) where the substance to be detected is captured by
the capturer 60 is defined by the second frame 50, the second frame
50 serves as a mark, and position adjustment of the irradiation
position of the excitation light .alpha. in the SPFS apparatus 100
is facilitated.
[0038] The shape and size of the second through-hole 51 are not
particularly limited and can be set appropriately according to the
application. Examples of the shape of the second through-hole 51
include a cylindrical trapezoidal shape, a cylindrical shape, an
elliptic cylindrical shape, a polygonal columnar shape, and
combinations thereof. From the viewpoint of making it easier to
remove the liquid in the well 53 and from the viewpoint of ease of
processing the second frame 50, it is preferable that the shape of
the second through-hole 51 is a cylindrical shape or an elliptic
cylindrical shape. In the embodiment, the shape of the second
through-hole 51 is a combination of a cylindrical shape and a
cylindrical trapezoidal shape.
[0039] As illustrated in FIG. 2B, the depth h2 of the second
through-hole 51 is preferably higher than the height h1 of the
first through-hole 41. Therefore, it is possible to prevent the
liquid from scattering out of the well 53 in a case where the
liquid is fed into the well 53. The height of the second
through-hole 51 can be appropriately set according to the height
and amount of the fed liquid bouncing back from the metal film 30
and the inner wall surface of the second through-hole 51.
[0040] The outer shape of the second frame 50 is not particularly
limited. For example, the outer shape of the second frame 50 in a
plan view is a circular shape, a quadrilateral shape, or the like.
In the embodiment, the outer shape of the second frame 50 in a plan
view is a circular shape. In addition, it is preferable that the
second frame 50 has a tapered portion where the cross-sectional
area of the second through-hole 51 increases in the direction
perpendicular to the height direction of the second through-hole 51
as the distance from the prism 20 increases. Therefore, it is
possible to easily supply the liquid into the second through-hole
51 (the well 53). In the embodiment, the second frame 50 has a
tapered portion 52.
[0041] In a case where the second frame 50 has the tapered portion
52, it is further preferable that the inner wall surface of the
tapered portion 52 is inclined so that the light beam emitted from
the center of the surface (reaction field) of the metal film 30
exposed in the second through-hole 51 and emitted at an emitting
angle of 20.degree. or less is not blocked. Therefore, it is
possible to prevent the fluorescence light .gamma. emitted from the
fluorescent substance labeling the substance to be detected from
being blocked, so that it is possible to detect the substance to be
detected with high accuracy.
[0042] It is preferable that the inner wall surface of the second
through-hole 51 is subjected to a blocking treatment. Therefore, it
is possible to suppress non-specific binding of the substance to be
detected to the inner wall surface of the second through-hole 51,
so that it is possible to improve the detection efficiency of the
substance to be detected.
[0043] In addition, the second frame 50 may have other elements as
required. For example, the second frame 50 may have a reagent
holder for accommodating the reagent.
[0044] The shape and color of the inner wall surface of the second
frame 50 can be appropriately set as required. For example, in a
case where a large amount of noise light is included in the
detected light, the inner wall surface of the second frame 50 may
be configured to be black from the viewpoint of absorbing excessive
noise light. In addition, in a case where the noise light has
directionality, the inner wall surface of the second frame 50 may
be configured to have minute irregularities from the viewpoint of
dispersing noise light to reduce noise. In addition, the inner wall
surface of the second frame 50 may be configured to be white, or
the inner wall surface of the second frame 50 may be configured to
be a mirror surface from the viewpoint of reflecting the
fluorescence light .gamma. emitted from the fluorescent substance
labeling the substance to be detected toward the light receiving
unit 130.
[0045] Examples of the material of the second frame 50 include
resin and glass. For example, the second frame 50 may be a resin
film. Therefore, the manufacturing becomes easy, the manufacturing
cost can be reduced, and the sensing chip 10 can be
miniaturized.
[0046] The second frame 50 is bonded to the metal film 30 or the
prism 20 by, for example, bonding with double-sided tape or
adhesive, laser welding, ultrasonic welding, crimping using a clamp
member, or the like. In addition, the second frame 50 may be bonded
to the first frame 40.
[0047] As illustrated in FIG. 1, the excitation light .alpha.
guided to the prism 20 is incident on the incidence surface 21 into
the prism 20. The excitation light .alpha. incident into the prism
20 is incident on the interface (film formation surface 22) between
the prism 20 and the metal film 30 so that a total reflection angle
(the angle at which surface plasmon resonance occurs) is formed.
The reflected light reflected at the interface is emitted to the
outside of the prism 20 from the light emission surface 23 (not
illustrated). At this time, the excitation light .alpha. is
incident on the interface at the angle at which the surface plasmon
resonance occurs, so that the plasmon scattered light .beta., the
fluorescence light .gamma. from the fluorescent substance, and the
like are emitted above the sensing chip 10 from the reaction
field.
[0048] (Method of Manufacturing Sensing Chip)
[0049] Next, an example of a method of manufacturing the sensing
chip 10 according to the embodiment will be described. FIG. 3 is a
flowchart illustrating an example of processes of manufacturing the
sensing chip 10 according to the embodiment. FIGS. 4A to 4D are
schematic diagrams illustrating an example of the processes of
manufacturing the sensing chip 10 according to the embodiment.
[0050] For example, the sensing chip 10 according to the embodiment
can be manufactured by performing a first step (step S10) of
preparing the prism 20, a second step (step S20) of arranging the
first frame 40 on the prism 20 (metal film 30)), a third step (step
S30) of immobilizing the capturer 60 on the metal film 30 exposed
in the first through-hole 41, and a fourth step (step S40) of
arranging the second frame 50 in the first through-hole 41.
[0051] 1) First Step
[0052] In the first step, as illustrated in FIG. 4A, the prism 20
(support) having the incidence surface 21, the film formation
surface 22, and the light emission surface 23 and having the metal
film 30 formed on one surface (film formation surface 22) is
prepared (step S10). First, the prism 20 is molded into a desired
shape. A method of molding the prism 20 is not particularly
limited, and for example, the prism 20 may be molded by a die
molding method. Next, the metal film 30 is formed on the film
formation surface 22 of the prism 20. Examples of a method of
forming the metal film 30 include sputtering, vapor deposition, and
plating. Alternatively, the prism 20 on which the metal film 30 has
already been formed may be purchased.
[0053] 2) Second Step
[0054] In the second step, as illustrated in FIG. 4B, the first
frame 40 having the first through-hole 41 is arranged on the metal
film 30 so that one opening of the first through-hole 41 is closed
(step S20). A method of forming the first through-hole 41 in the
first frame 40 is not particularly limited, and the first
through-hole 41 may be formed by, for example, a die molding
method, a cutting process, or the like.
[0055] 3) Third Step
[0056] In the third step, as illustrated in FIG. 4C, the capturer
60 is immobilized on the metal film 30 exposed in the first
through-hole 41 (step S30). Specifically, a liquid containing the
capturer 60 is fed into the first through-hole 41 of the first
frame 40 arranged on the metal film 30. Therefore, the capturer 60
is immobilized on at least a portion of the inner wall surface of
the first through-hole 41 of the first frame 40 which is in contact
with the fed liquid and the surface of the metal film 30 exposed in
the first through-hole 41.
[0057] In addition, a method of immobilizing the capturer 60 on the
metal film 30 is not particularly limited. For example, a
self-assembled monolayer (hereinafter, referred to as "SAM") or a
polymer membrane to which the capturer 60 is bonded may be formed
on the metal film 30. Examples of the SAM include membranes formed
with substituted aliphatic thiols such as
HOOC--(CH.sub.2).sub.11--SH. Examples of materials constituting the
polymer membrane include polyethylene glycol and MPC polymers. In
addition, a polymer having a reactive group (or a functional group
convertible to a reactive group) capable of being bonded to the
capturer 60 may be immobilized on the metal film 30, and the
capturer 60 may be bonded to the polymer.
[0058] 4) Fourth Step
[0059] In the fourth step, after removing the liquid containing the
capturer 60 from above the metal film 30, as illustrated in FIG.
4D, the second frame 50 having the second through-hole 51 is
arranged on the metal film 30 in the first through-hole 41 so that
one opening of the second through-hole 51 is closed (step S40).
Typically, after removing the liquid containing the capturer 60,
before arranging the second frame 50, the inside of the first
through-hole 41 is cleaned with a buffer solution or the like. In
addition, although not illustrated in FIG. 4D, even if the second
frame 50 is arranged in the first through-hole 41, the capturer 60
is immobilized on a portion of the inner wall surface of the first
through-hole 41 of the first frame 40 and the surface of the metal
film 30. The second through-hole 51 can be formed by, for example,
the same method as the first through-hole 41.
[0060] Through the above-mentioned steps, the sensing chip 10 used
for detecting the substance to be detected contained in the
specimen can be manufactured.
[0061] As an arbitrary step, a step of performing a blocking
treatment on the second frame 50 may be further included. In the
blocking treatment, for example, a liquid containing a blocking
agent may be brought into contact with the inner wall surface of
the second frame 50. The blocking treatment may be performed in
advance before the second frame 50 is arranged on the metal film 30
or after the second frame 50 is arranged on the metal film 30.
Examples of blocking agents include high molecular weight compounds
such as casein, skim milk, albumin (including bovine serum
albumin), gelatin, polyethylene glycol, and the like, low molecular
weight compounds such as phospholipids, ethylenediamine and
acetonitrile, and the like. These blocking agents may be used alone
or in combination of two or more.
[0062] In addition, as an arbitrary step, a step of protecting the
capturer 60 with a moisturizing agent may be included to prevent
the capturer 60 from drying out.
[0063] In the sensing chip 10 according to the embodiment
manufactured in this manner, the capturer 60 is also immobilized on
a portion of the inner wall surface of the first through-hole 41 of
the first frame 40. However, since the second frame 50 arranged in
the inside of the first through-hole 41 defines the well 53, the
liquid introduced into the well 53 will not be in contact with the
inner wall surface of the first through-hole 41. In addition, the
capturer 60 is not immobilized on the inner wall surface of the
second through-hole 51 of the second frame 50. Therefore, in a case
where a specimen is introduced into the well 53, the substance to
be detected contained in the specimen is efficiently captured by
the capturer 60 immobilized on the bottom surface of the well
53.
[0064] (Configuration of SPFS Apparatus)
[0065] Next, each component of the SPFS apparatus 100 will be
described. As described above, the SPFS apparatus 100 is configured
to include the chip holder 110, the light irradiation unit 120, the
light receiving unit 130, and the control unit 140 (refer to FIG.
1).
[0066] The chip holder 110 holds the sensing chip 10 at a
predetermined position. The sensing chip 10 is irradiated with the
excitation light .alpha. from the light irradiation unit 120 in the
state where the sensing chip is held by the chip holder 110.
[0067] The light irradiation unit 120 irradiates the incidence
surface 21 of the prism 20 of the sensing chip 10 held by the chip
holder 110 with the excitation light .alpha. (single mode laser
light). More specifically, the light source unit 121 emits the
excitation light .alpha. to a region corresponding to the well 53
on the back surface of the metal film 30 so that a total reflection
angle is formed.
[0068] The light irradiation unit 120 is configured to include a
light source unit 121 that emits the excitation light .alpha., an
angle adjustment unit 122 that adjusts the incidence angle of the
excitation light .alpha. to the interface (film formation surface
22) between the prism 20 and the metal film 30, and a light source
control unit 123 that controls various devices included in the
light source unit 121.
[0069] The light source unit 121 emits the excitation light
.alpha.. For example, the light source unit 121 has a light source
of the excitation light .alpha., a beam shaping optical system, an
APC mechanism, and a temperature adjustment mechanism (all the
components are not illustrated).
[0070] The type of the light source is not particularly limited.
Examples of type of light source include laser diodes (LD), light
emitting diodes, mercury lamps, and other laser light sources.
[0071] In a case where the excitation light .alpha. emitted from
the light source is not a beam, the excitation light .alpha.
emitted from the light source is converted into a beam by a lens, a
mirror, a slit, or the like. In addition, in a case where the
excitation light .alpha. emitted from the light source is not
monochromatic light, the excitation light .alpha. emitted from the
light source is converted into monochromatic light by a diffraction
grating or the like. Furthermore, in a case where the excitation
light .alpha. emitted from the light source is not linearly
polarized light, the excitation light .alpha. emitted from the
light source is converted into linearly polarized light by a
polarizer or the like.
[0072] The beam shaping optical system is configured to include,
for example, a collimator, a band pass filter, a linear polarizing
filter, a half wave plate, a slit, a zoom means, and the like. The
beam shaping optical system may include all or some of the
components.
[0073] The collimator collimates the excitation light
.alpha.emitted from the light source.
[0074] The band pass filter converts the excitation light .alpha.
emitted from the light source into narrow band light having only
the center wavelength. This is because the excitation light .alpha.
from the light source has a slight wavelength distribution
width.
[0075] The linear polarizing filter converts the excitation light
.alpha. emitted from the light source into completely linearly
polarized light. The half-wave plate adjusts the polarization
direction of the excitation light .alpha. so that the P-wave
component light is incident on the metal film 30. The slit and the
zooming means adjust the beam diameter and contour shape of the
excitation light .alpha. so that the shape of the irradiation spot
on the back surface of the metal film 30 becomes a circle having a
predetermined size.
[0076] The APC mechanism controls the light source so that the
output of the light source is constant. More specifically, the APC
mechanism detects the light amount of light branched from the
excitation light .alpha. with a photodiode (not illustrated) or the
like. Then, the APC mechanism controls the output of the light
source to be constant by controlling the input energy with a
recursion circuit.
[0077] The temperature adjustment mechanism is, for example, a
heater or a Peltier element. The wavelength and energy of the light
emitted from the light source may vary depending on the
temperature. Therefore, by maintaining the temperature of the light
source constant by the temperature adjustment mechanism, the
wavelength and the energy of the light emitted from the light
source are controlled to be constant.
[0078] The angle adjustment unit 122 adjusts the incidence angle of
the excitation light .alpha. to the metal film 30 (the interface
(film formation surface 22) between the prism 20 and the metal film
30). The angle adjustment unit 122 rotates the optical axis of the
excitation light .alpha. and the chip holder 110 relative to each
other in order to irradiate a predetermined position of the metal
film 30 (film formation surface 22) with the excitation light
.alpha. at a predetermined incidence angle. In the embodiment, the
angle adjustment unit 122 rotates the light source unit 121 around
an axis (an axis perpendicular to the paper surface of FIG. 1)
perpendicular to the optical axis of the excitation light .alpha.
on the metal film 30.
[0079] The light source control unit 123 controls various devices
included in the light source unit 121 to adjust the power of the
excitation light .alpha. from the light source unit 121, the
irradiation time, and the like. The light source control unit 123
is configured with, for example, a well-known computer,
microcomputer, or the like including an arithmetic device, a
control device, a storage device, an input device, and an output
device.
[0080] The light receiving unit 130 is arranged so as to face a
surface of the metal film 30 of the sensing chip 10 held by the
chip holder 110, which does not face the prism 20. The light
receiving unit 130 detects the light (plasmon scattered light
.beta. or fluorescence light .gamma.) emitted from the metal film
30 in the second through-hole 51. The light receiving unit 130 is
configured to include a first lens 132, an optical filter 133, a
second lens 134, and a light receiving sensor 135 which are
arranged in the light receiving optical system unit 131, a position
switching mechanism 136, and a light sensor control unit 137.
[0081] The first lens 132 is, for example, a condenser lens and
condenses the light emitted from the metal film 30. The second lens
134 is, for example, an imaging lens and focuses the light
condensed by the first lens 132 on the light receiving surface of
the light receiving sensor 135. The optical paths between the two
lenses are substantially parallel.
[0082] The optical filter 133 is arranged between the first lens
132 and the second lens 134. The optical filter 133 transmits only
the fluorescence light component of the incident light, and removes
the excitation light component (plasmon scattered light ). By
removing the excitation light component by the optical filter 133,
it is possible to detect the fluorescence light .gamma. with a high
S/N ratio. Examples of types of the optical filter 133 include an
excitation light reflection filter, a short wavelength cut filter,
and a band pass filter.
[0083] The light receiving sensor 135 detects the plasmon scattered
light .beta. and the fluorescence light .gamma. emitted from the
sensing chip 10. The type of the light receiving sensor 135 is not
particularly limited as long as the above object can be achieved,
but it is preferable that the variation of the detection value is
small even if the received light amount increases. The light
receiving sensor 135 is, for example, a photodiode (PD).
[0084] The position switching mechanism 136 switches the position
of the optical filter 133 onto the optical path or out of the
optical path in the light receiving optical system unit 131.
Specifically, when the optical blank value or the fluorescence
value is to be measured, the optical filter 133 is arranged on the
optical path in the light receiving optical system unit 131, and
when the light receiving sensor 135 is to detect the plasmon
scattered light .beta., the optical filter 133 is arranged out of
the optical path.
[0085] The light sensor control unit 137 controls detecting the
output value of the light receiving sensor 135 and managing the
sensitivity of the light receiving sensor 135 based on the detected
output value and controls the sensitivity of the light receiving
sensor 135 to obtain an appropriate output value. The light sensor
control unit 137 is configured with, for example, a well-known
computer, microcomputer, or the like including an arithmetic
device, a control device, a storage device, an input device, and an
output device.
[0086] The control unit 140 controls the angle adjustment unit 122,
the light source control unit 123, the position switching mechanism
136, and the light sensor control unit 137. The control unit 140
also functions as a processing unit for calculating a signal value
indicating the presence or amount of the substance to be detected
based on a detection result of the light receiving sensor 135. The
control unit 140 is configured with, for example, a well-known
computer, microcomputer, or the like including an arithmetic
device, a control device, a storage device, an input device, and an
output device.
[0087] [Operation of SPFS Apparatus]
[0088] Next, operations of the SPFS apparatus 100 using the sensing
chip 10 will be described. FIG. 5 is a flowchart illustrating an
example of the operation procedure of the SPFS apparatus.
[0089] First, detection is prepared (step S110). Specifically, the
sensing chip 10 is installed in the chip holder 110 of the SPFS
apparatus 100. In a case where a moisturizing agent is present in
the well 53 of the sensing chip 10, the interior of the well 53 is
cleaned so as to remove the moisturizing agent so that the capturer
60 can appropriately capture the substance to be detected.
[0090] Next, the enhancement angle is determined (step S120).
Specifically, while irradiating the predetermined position of the
metal film 30 (film formation surface 22) with the excitation light
.alpha., the incidence angle of the excitation light .alpha. on the
metal film 30 (film formation surface 22) is scanned to determine
an optimum incidence angle. The control unit 140 controls the light
source control unit 123 and the angle adjustment unit 122 to scan
the incidence angle of the excitation light .alpha. to the metal
film 30 (film formation surface 22) while irradiating the
predetermined position of the metal film 30 (film formation surface
22) with the excitation light .alpha. from the light source unit
121. At this time, the control unit 140 controls the position
switching mechanism 136 to move the optical filter 133 to the
outside of the optical path of the light receiving optical system
unit 131. At the same time, the control unit 140 controls the light
sensor control unit 137 to detect the plasmon scattered light
.beta. with the light receiving sensor 135. The control unit 140
obtains data including the relationship between the incidence angle
of the excitation light .alpha. and the intensity of the plasmon
scattered light .beta.. Then, the control unit 140 analyzes the
data and determines the incidence angle (enhancement angle) at
which the intensity of the plasmon scattered light .beta. becomes
maximum. In addition, although the enhancement angle is determined
by the material and shape of the prism 20, the thickness of the
metal film 30, the refractive index of the liquid in the well 53,
and the like, the enhancement angle varies slightly depending on
various factors such as the type and amount of the capturer 60 and
the shape error of the prism 20. Therefore, it is preferable to
determine the enhancement angle each time the detection is
performed. The enhancement angle is determined on the order of
about 0.1.degree..
[0091] Next, the incidence angle of the excitation light .alpha. to
the metal film 30 (film formation surface 22) is set to be the
enhancement angle determined in step S120 (step S130).
Specifically, the control unit 140 controls the angle adjustment
unit 122 to set the incidence angle of the excitation light .alpha.
to the metal film 30 (film formation surface 22) to be the
enhancement angle. In the subsequent steps, the incidence angle of
the excitation light .alpha. to the metal film 30 (film formation
surface 22) is the enhancement angle.
[0092] Next, in the state where no fluorescent substance is present
on the metal film 30, detection of light including light having the
same wavelength as the fluorescence light .gamma. is performed, and
the optical blank value is measured (step S140). Herein, the
"optical blank value" denotes the light amount of background light
emitted above the sensing chip 10. This background light is mainly
caused by auto-fluorescence light emitted from the sensing chip 10
(prism 20) and external light in the irradiation with the
excitation light .alpha..
[0093] Specifically, the control unit 140 controls the position
switching mechanism 136 to move the optical filter 133 onto the
optical path of the light receiving optical system unit 131. Next,
the control unit 140 controls the light source control unit 123 to
irradiate the metal film 30 with the excitation light .alpha.
through the prism 20 from the light source unit 121 so that the
surface plasmon resonance occurs in the metal film 30 in the state
where there is no fluorescent substance on the metal film 30. At
the same time, the control unit 140 controls the light sensor
control unit 137 to detect the light emitted from the sensing chip
10 by the light receiving sensor 135 and obtains the optical blank
value. The measured optical blank value is transmitted to and
stored in the control unit (processing unit) 140.
[0094] Subsequently, the substance to be detected in the specimen
and the capturer 60 are allowed to react with each other (primary
reaction; step S150). Specifically, the specimen is injected into
the well 53, and the specimen and the capturer 60 are brought into
contact with each other. In a case where a substance to be detected
is present in the specimen, at least a portion of the substance to
be detected is captured by the capturer 60. At this time, the
substance to be detected is appropriately captured by the capturer
60 immobilized on the bottom surface of the well 53. The capturer
60 is also immobilized on a portion of the inner wall surface of
the first through-hole 41 of the first frame 40, but contact
between the capturer 60 immobilized at a position other than the
bottom surface of the well 53 and the specimen is hindered by the
second frame 50. In addition, on the inner wall surface of the
second through-hole 51 of the second frame 50, the capturer 60 is
not immobilized. Therefore, the substance to be detected is
efficiently captured by the capturer 60 immobilized on the bottom
surface of the well 53. After that, the interior of the well 53 is
cleaned with a buffer solution or the like to remove substances not
captured by the capturer 60. The type of the specimen is not
particularly limited. Examples of the specimens include body fluids
such as blood and serum, plasma, urine, nostrils, saliva, semen and
diluents thereof.
[0095] Subsequently, the substance to be detected captured by the
capturer 60 is labeled with a fluorescent substance (secondary
reaction; step S160). Specifically, a fluorescent labeling solution
is fed into the well 53. The fluorescent labeling solution is, for
example, a buffer solution containing an antibody (secondary
antibody) labeled with the fluorescent substance. When the
fluorescent labeling solution is fed into the well 53, the
fluorescent labeling solution comes into contact with the substance
to be detected captured by the capturer 60, so that the substance
to be detected is labeled with the fluorescent substance.
Thereafter, the inside of the well 53 is cleaned with a buffer
solution or the like to remove free fluorescent substances and the
like.
[0096] Next, in the state where the substance to be detected
labeled with a fluorescent substance is present in the well 53, by
irradiating the metal film 30 (film formation surface 22) with the
excitation light .alpha. and detecting the fluorescence light y
emitted from the fluorescent substance that labels the substance to
be detected in the reaction field, the fluorescence value is
measured (step S170). Specifically, the control unit 140 controls
the light source control unit 123 to allow the excitation light
.alpha. to be emitted from the light source unit 121 to the metal
film 30 through the prism 20 so that the surface plasmon resonance
occurs in the metal film 30 in the state where the substance to be
detected labeled with the fluorescent substance is present on the
metal film 30. At the same time, the control unit 140 controls the
light sensor control unit 137 to detect the fluorescence light
.gamma. emitted from the fluorescent substance labeling the
substance to be detected with the light receiving sensor 135. The
measured fluorescence value is transmitted to the control unit
(processing unit) 140 and stored.
[0097] Finally, a signal value indicating the presence or amount of
the substance to be detected is calculated (step S180).
Specifically, the control unit 140 calculates a signal value
correlating to the amount of the substance to be detected by
subtracting the optical blank value obtained in step S140 from the
fluorescence value obtained in step S170. The signal value can be
reduced into the amount or concentration of the substance to be
detected by a calibration curve prepared in advance.
[0098] By performing the above-described operation procedure, it is
possible to detect the substance to be detected with high accuracy
by using the sensing chip 10 according to the embodiment.
[0099] In addition, the order of steps S120 to S180 is not limited
to the above order. For example, after performing the primary
reaction (step S150), the determining of the enhancement angle
(step S120), the setting of the incidence angle to the enhancement
angle (step S130) and the measuring of the optical blank value
(step S140) may be performed.
[0100] (Effect)
[0101] As described above, by using the sensing chip 10 according
to the embodiment, it is possible to suppress the adhesion of the
substance to be detected to the inner wall surface of the well 53
and to reduce the number of substances to be detected that are not
detected. Therefore, it is possible to detect the substance to be
detected in the specimen with high accuracy.
[0102] In addition, in the above-described embodiment, the sensing
chip 10 having the first frame 40 of which the shape of the first
through-hole 41 is a cylindrical shape has been described. However,
the shape of the first frame 40 in the sensing chip 10 according to
the embodiment is not limited thereto. FIGS. 6A and 6B are diagrams
illustrating a configuration of a sensing chip 10' according to
Modified Example 1. FIG. 6A is a plan diagram of the sensing chip
10', and FIG. 6B is a cross-sectional diagram taken along the line
B-B in FIG. 6A. As illustrated in FIGS. 6A and 6B, the first frame
40' may have a tapered portion 42' so that, as the first frame 40'
is away from the prism 20, the cross-sectional area of the first
through-hole 41' in the direction perpendicular to the height
direction of the first through-hole 41' increases. Therefore, it is
possible to easily feed a liquid (for example, a liquid containing
the capturer 60, a cleaning liquid, or the like) in the first
through-hole 41'. In addition, in the sensing chip 10' according to
Modified Example 1, a second frame 50' may be arranged so as to be
in contact with the first frame 40' without a gap or may be
arranged to be separated from the first frame. In the sensing chip
10' according to Modified Example 1, the shape of the second frame
50' (the second through-hole 51') is formed so as to be in contact
with the first frame 40' without a gap.
[0103] In addition, in the above-described embodiment, the sensing
chip 10 having the second frame 50 including the tapered portion
has been described. However, the shape of the second frame of the
sensing chip 10 according to the present invention is not limited
thereto. FIGS. 7A and 7B are diagrams illustrating a configuration
of a sensing chip 10'' according to Modified Example 2. FIG. 7A is
a plan diagram of the sensing chip 10'', and FIG. 7B is a
cross-sectional diagram taken along the line B-B in FIG. 7A. As
illustrated in FIGS. 7A and 7B, a second through-hole 51'' of a
second frame 50'' may have a cylindrical shape. In the sensing chip
10'' according to Modified Example 2, the shape of a well 53'' is a
cylindrical shape. Also in this case, the same effects as the
above-described embodiment can be obtained by manufacturing the
sensing chip 10'' similarly to the above-described embodiment.
[0104] In addition, in the above-described embodiment, a case where
the sensing chips 10, 10', and 10'' are used for detecting the
substance to be detected by using the SPFS has been described.
However, the sensing chip according to the present invention is not
limited thereto, and the sensing chip can be used for other
immunoassays. In this case, a support such as a glass substrate may
be used instead of the prism 20 having the metal film 30 formed on
one surface.
[0105] This application claims priority based on Japanese Patent
Application No. 2015-087588 filed on Apr. 22, 2015. The entire
contents described in the application specification and drawings
are incorporated herein by reference.
INDUSTRIAL APPLICABILITY
[0106] According to a sensing chip of the present invention, since
a substance to be detected can be detected with high sensitivity
and high accuracy, the sensing chip is useful for, for example,
clinical examinations.
REFERENCE SIGNS LIST
[0107] 10, 10', 10'' sensing chip
[0108] 20 prism
[0109] 21 incidence surface
[0110] 22 film formation surface
[0111] 23 light emission surface
[0112] 30 metal film
[0113] 40, 40' first frame
[0114] 41, 41' first through-hole
[0115] 50, 50', 50'' second frame
[0116] 51, 51', 51'' second through-hole
[0117] 42', 52 tapered portion
[0118] 43 reagent holder
[0119] 53, 53'' well
[0120] 60 capturer
[0121] 100 surface plasmon resonance fluorescence analysis
apparatus (SPFS apparatus)
[0122] 110 chip holder
[0123] 120 light irradiation unit
[0124] 121 light source unit
[0125] 122 angle adjustment unit
[0126] 123 light source control unit
[0127] 130 light receiving unit
[0128] 131 light receiving optical system unit
[0129] 132 first lens
[0130] 133 optical filter
[0131] 134 second lens
[0132] 135 light receiving sensor
[0133] 136 position switching mechanism
[0134] 137 light sensor control unit
[0135] 140 control unit (processing unit)
[0136] .alpha. excitation light
[0137] .beta. plasmon scattered light
[0138] .gamma. fluorescence light
[0139] h1 height of first through-hole
[0140] h2 height of second through-hole
* * * * *