U.S. patent application number 14/467652 was filed with the patent office on 2014-12-11 for chip for optical analysis.
The applicant listed for this patent is NIPPON TELEGRAPH AND TELEPHONE CORPORATION. Invention is credited to Serge Camou, Tsuneyuki Haga, Tsutomu Horiuchi, Yuzuru Iwasaki, Toru Miura, Michiko Seyama, Jyunichi Takahashi.
Application Number | 20140363337 14/467652 |
Document ID | / |
Family ID | 38831694 |
Filed Date | 2014-12-11 |
United States Patent
Application |
20140363337 |
Kind Code |
A1 |
Seyama; Michiko ; et
al. |
December 11, 2014 |
CHIP FOR OPTICAL ANALYSIS
Abstract
The present invention relates to a chip for optical analysis. In
particular, the present invention relates to an optical sensor
handling a liquid sample, which is a chip for analysis that can be
used for selectively measuring a biologically-relevant substance or
a chemical substance such as an environmental pollutant or a health
affecting substance in a liquid to be measured. The chip for
optical analysis of the present invention is characterized in that
(1) an adsorption region (filter region) is provided between a
sample introduction section and the observation section in a
passage of the chip for analysis, (2) a bypass passage is provided
in the passage (main passage) of the chip for analysis, and a time
lag is generated between samples passed through the main passage
and passed through a bypass passage, and (3) a measurement region
and a reference region are provided in the observation section of
the chip for analysis. In the present invention, the aspects (1) to
(3) can be achieved individually or two or more thereof can be
combined
Inventors: |
Seyama; Michiko;
(Atsugi-shi, JP) ; Camou; Serge; (Atsugi-shi,
JP) ; Iwasaki; Yuzuru; (Atsugi-shi, JP) ;
Miura; Toru; (Atsugi-shi, JP) ; Takahashi;
Jyunichi; (Atsugi-shi, JP) ; Haga; Tsuneyuki;
(Atsugi-shi, JP) ; Horiuchi; Tsutomu; (Atsugi-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NIPPON TELEGRAPH AND TELEPHONE CORPORATION |
Tokyo |
|
JP |
|
|
Family ID: |
38831694 |
Appl. No.: |
14/467652 |
Filed: |
August 25, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12304112 |
Jun 10, 2010 |
8859267 |
|
|
PCT/JP2007/061757 |
Jun 11, 2007 |
|
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14467652 |
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Current U.S.
Class: |
422/69 |
Current CPC
Class: |
B01L 2300/0861 20130101;
B01L 2300/0864 20130101; B01L 3/502715 20130101; B01L 2300/0825
20130101; B01L 2300/069 20130101; G01N 21/553 20130101; B01L
2300/0645 20130101; G01N 33/54373 20130101; B01L 2200/148 20130101;
B01L 2400/084 20130101; B01L 2300/0816 20130101 |
Class at
Publication: |
422/69 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 12, 2006 |
JP |
2006-162879 |
Jun 14, 2006 |
JP |
2006-165172 |
Jun 26, 2006 |
JP |
2006-175682 |
Claims
1. A chip for optical analysis comprising a substrate, a metal thin
film on the substrate, and an observation section on the metal foil
membrane, wherein the observation section comprises a measurement
region and a reference region, and on the measurement region, a
first substance having molecular selectivity that selectively
interacts with a specific molecule is immobilized, and on the
reference region, a second substance having molecular selectivity
that is different only in selectivity to the specific molecule with
which the first substance having molecular selectivity interacts
and is comparable to the first substance having molecular
selectivity in other characteristics is immobilized, and the first
substance having molecular selectivity and the second substance
having molecular selectivity are immobilized on the substrate by
the same method.
2. The chip for optical analysis according to claim 1, wherein the
first substance having molecular selectivity and the second
substance having molecular selectivity are antibodies or antigens
of the same kinds.
3. The chip for optical analysis according to claim 1, wherein the
second substance having molecular selectivity is obtained by
inactivating the first substance having molecular selectivity, and
inactivation is achieved by irradiation with a high energy-ray
selected from an X-ray, a gamma (.gamma.)-ray, and an electron
beam, heat treatment, electrochemical oxidation or reduction, or
contact with an acidic or alkaline buffer.
4. The chip for optical analysis according to claim 1, wherein the
first substance having molecular selectivity and the second
substance having molecular selectivity are DNA, and the second
substance having molecular selectivity is obtained by replacing 10%
or less of nucleotides constituting the first substance having
molecular selectivity.
5. The chip for optical analysis according to claim 1, wherein a
plurality of observation sections each comprising the measurement
region and the reference region as one set are provided in the
passage on the substrate, and first substances having molecular
selectivity different from each other are immobilized on the
measurement region of the observation sections, so that each
observation section can detect a different molecule.
Description
TECHNICAL FIELD
[0001] The present invention relates to a chip for optical
analysis. In particular, the present invention relates to a chip
for analysis, which can be used for an optical sensor for handling
a liquid sample that selectively measures chemical substances such
as a biologically-relevant substance or an environmental pollutant
or a health affecting substance in a liquid to be measured. The
chip for analysis of the present invention can use a substance
having molecular selectivity. Furthermore, the chip for analysis of
the present invention measures a real sample containing impurities,
with higher measurement sensitivity and higher precision in
measurement. The chip for analysis of the present invention is
intended for a simpler operation in measurement and higher
sensitivity and higher precision in measurement.
BACKGROUND ART
[0002] The method for measuring a chemical substance in a liquid
sample include for example electric and optical measurement methods
in which a substance having molecular selectivity is immobilized
beforehand, a liquid sample is allowed to flow thereon, and a bound
molecule is selectively detected.
[0003] Optical methods involve techniques using a probe molecule to
which a fluorescent substance or a light-absorbing substance is
immobilized beforehand. In addition, methods using a total
reflection optical system are available by which binding of a
molecule and an immobilized substance having molecular selectivity
can be directly observed. These methods are known as methods in
which an excited evanescent wave is used as a probe light, and
binding in the vicinity of the surface is directly measured with
high sensitivity. Most commonly used method among the total
reflection-type optical systems is Surface Plasmon Resonance (SPR)
measurement method, which utilizes a sample's absorption of a
surface plasmon excited by total reflection of a metal thin film
formed on a substrate surface, and total reflection-type
attenuation measurement method, which utilizes a sample's
absorption of an evanescent wave excited by total reflection due to
a difference between a substrate surface and a sample in a
refractive index.
[0004] To measure a liquid sample with a total reflection optical
system, means of forming a passage on a substrate and immobilizing
a probe molecule in the passage beforehand or forming a passage on
an immobilized probe molecule is taken. Then, a liquid sample is
passed through this passage. Based on an interaction between the
immobilized probe molecule and a substance to be measured in a
sample, whether the substance to be measured is contained in the
sample can be determined, and the substance to be measured can be
further quantified using the above-mentioned total reflection
optical system (for example, the amount of a specific chemical
substance contained in a liquid can be determined from the
condition of a reflected light of a measurement light irradiated on
the above-mentioned passage).
[0005] Furthermore, measurement using a total reflection optical
system and a substance having molecular selectivity appears to be
more suitable for short-time low-cost screening that is also
suitably used in a field test than high-cost, high-precision, and
high-sensitivity analyses represented by chromatography. A simple
device for total reflection optical measurement has already been
developed and described in, for example, Patent Document 1.
[0006] [Patent Document 1] Japanese Patent Laid-Open No.
2002-214131
DISCLOSURE OF THE INVENTION
[0007] To conduct a high-sensitivity analysis in optical
measurement, preparation of a reference sample, which does not
contain a measurement object and serves as a reference, is
required. While it is possible to separately prepare a sample not
containing a substance to be measured as a reference for a real
sample, which contains a substance to be measured, a sample
obtained by removing the substance to be measured from the real
sample itself is considered to be ideal as a reference. From this
viewpoint, a reference sample obtained by removing a substance to
be measured from a real sample is prepared and used as a reference
in some cases. However, when this reference sample is prepared, a
complex pretreatment operation may be required, and such necessity
of a pretreatment operation is not desirable in on-site or simple
measurement.
[0008] Furthermore, for example, when a sample to be actually
measured such as serum, blood, urine, an animal body fluid other
than these, environmental water, or a liquid collected from a plant
is a liquid sample, many impurities exist in such a liquid sample
in addition to the substance to be measured. When total reflection
optical measurement is performed, it is necessary to eliminate
effects of such impurities and selectively detect the substance to
be measured. In consideration of on-site measurement, such
elimination treatment of impurities is not desirable either.
[0009] Such measurement by a total reflection optical system in
which a probe molecule is immobilized on a substrate beforehand as
described above suffers from a problem that many impurities are
adsorbed or accumulated in a space on a substrate in which a
measurement region is provided. Conventionally, when such
accumulation of impurities occurs due to adsorption, effects of
impurities are suppressed to some extent by using a region in which
a probe molecule is not immobilized as a reference region,
performing differential measurement between a measurement region
and the reference region, and deducting changes other than a
reaction by a probe molecule. However, when a measurement object
having a low concentration is to be detected, adsorption and
accumulation of impurities in the reference region in which a probe
molecule does not exist often differ from those in the measurement
region. Therefore, a problem arises that, even if a difference
between the reference region and the measurement region is
deducted, effects of impurities cannot be eliminated, a baseline
for measurement changes, and the baseline changes more greatly than
a signal to be detected.
[0010] Furthermore, devices by which a change in the refractive
index of a sample is read from a change in the resonance angle are
commonly used in surface plasmon resonance measurement methods.
However, since the refractive index of a liquid sample is greater
than that of a gas, the resonance angle may change about 15 to 20
degrees after introduction of a sample. Such a great change in the
resonance angle can be detected with an analytical device for a
laboratory, which has a mobile detection system. In a simple device
without moving parts, however, it is impossible to detect a
resonance angle in a condition where a sample is not introduced,
and a signal cannot be obtained. On the contrary, a problem arises
that, even if a device has a mobile detection system, rapid
measurement is impossible because the detection operation requires
time.
[0011] An object of the present invention is to provide a chip for
optical analysis which can very easily and conveniently determine
how a measurement object is contained in a liquid to be measured by
preparing a reference sample from the liquid to be measured as a
reference.
[0012] Furthermore, another object of the present invention is to
provide a chip for optical analysis which can prepare a reference
sample in which a substance to be measured is free and selectively
detect a measurement object in the real sample only by introducing
a real sample into the chip for analysis.
[0013] Furthermore, another object of the present invention is to
provide a chip for analysis which can suppress a signal noise
generated from a difference in adsorption or accumulation of
impurities to a measurement region and a reference region in a
measurement method for detecting a substance to be measured based
on an interaction between a probe molecule immobilized on a
substrate and the substance to be measured, for example, a
measurement method using a total reflection optical system. Even
when a liquid sample to be measured by the chip for analysis of the
present invention contains many impurities, measurement using a
total reflection optical system can be achieved with higher
sensitivity and higher precision.
[0014] The present invention relates to a chip for optical
analysis. The chip for optical analysis of a first embodiment is a
chip for optical analysis provided with a substrate and a passage
on the substrate, wherein the passage comprises a sample
introduction section, an observation section, and an adsorption
region for adsorbing a substance to be measured between the sample
introduction section and the observation section.
[0015] The chip for optical analysis further comprises a passage
that bypasses the adsorption region from the sample introduction
section to the observation section, so that a sample passed through
the bypass passage can reach the observation section taking more
time than a sample passed through the adsorption region. In the
chip for optical analysis of the present invention, the observation
section can comprise at least a measurement region for detecting a
substance to be measured and a reference region.
[0016] On the measurement region, a material which is an antibody,
an antigen, an enzyme, an oligonucleotide, a DNA, an RNA, a
modified cyclodextrin, or a naturally occurring or synthesized
cyclic compound that functions as an ionophore can be
immobilized.
[0017] The adsorption region can contain protein A, dextran or a
modified product thereof, silica having an organic coating, or
alumina having an organic coating as an adsorbent.
[0018] The chip for optical analysis can further have a metal thin
film between the substrate and the passage. In the chip for optical
analysis, the passage and/or bypass passage may be a porous
membrane.
[0019] The chip for optical analysis of another embodiment
comprises a substrate that is transparent to a measurement light, a
chip main body joined to a surface of the substrate, an
introduction port formed in the chip main body for introducing a
liquid to be measured, a first passage through which a liquid to be
measured introduced through the introduction port passes, an
observation section arranged between the substrate and the chip
main body in the first passage and irradiated with the measurement
light, a metal thin film layer positioned at the observation
section and provided on a wall surface of the first passage, a
second passage branched from the first passage and merged again
into the first passage on the upstream side of the observation
section, an adsorption region for capturing a predetermined
component contained in a liquid to be measured, the adsorption
region positioned between a branching section and a merging section
with the second passage and incorporated in the first passage, and
means for generating a difference in time to reach the observation
section so that the liquid to be measured reaches the observation
section from the branching section through the first passage
earlier than reaching the observation section from the branching
section through the second passage.
[0020] The chip main body can further comprise a liquid absorbing
and holding section incorporated in this chip for optical analysis
and connected to the first passage on the downstream side of the
observation section to absorb the liquid to be measured passed
through the observation section.
[0021] The observation section can comprise at least a measurement
region for detecting a substance to be measured and a reference
region.
[0022] In the chip for optical analysis of this embodiment, the
means for generating a difference in time to reach the measurement
region may be (a) a hydrophilically treated layer formed on a wall
surface of the second passage, (b) at least one passage
resistance-increasing block projected from the surface of the
substrate facing the second passage into the second passage, (c) a
setting of the volume of the second passage larger than that of the
first passage from the branching section through the merging
section, or (d) a liquid collecting section provided halfway
through the second passage for collecting the liquid to be
measured.
[0023] Furthermore, in the chip for optical analysis of this
embodiment, a material having molecular selectivity may be
immobilized on the measurement region of the first passage. The
material having molecular selectivity can contain at least one
material selected from the group consisting of an antibody, an
antigen, an enzyme, an oligonucleoside, a ribonucleoside, and a
modified cyclodextrin compound. Furthermore, in the chip for
optical analysis of this embodiment, the adsorption region can
contain at least one adsorbent selected from a dextran gel using
protein A as a modifier, organic membrane-coated silica, and
organic membrane-coated alumina.
[0024] In each of the chips for optical analysis of the
embodiments, on the measurement region, a first substance having
molecular selectivity that selectively interacts with a specific
molecule is immobilized, and on the reference region, a second
substance having molecular selectivity which is different only in
selectivity to the specific molecule with which the first substance
having molecular selectivity interacts and is comparable to the
first substance having molecular selectivity in other
characteristics is immobilized, and the first substance having
molecular selectivity and the second substance having molecular
selectivity can be immobilized on the substrate by the same
method.
[0025] The chip for optical analysis of another embodiment of the
present invention is a chip for analysis, comprising a substrate, a
metal thin film on the substrate, and an observation section on the
metal thin film, wherein the observation section is provided with a
measurement region and a reference region, and on the measurement
region, a first substance having molecular selectivity that
selectively interacts with a specific molecule is immobilized, and
on the reference region, a second substance having molecular
selectivity that is different only in selectivity to the specific
molecule with which the first substance having molecular
selectivity interacts and is comparable to the first substance
having molecular selectivity in other characteristics is
immobilized, and the first substance having molecular selectivity
and the second substance having molecular selectivity can be
immobilized on the substrate by the same method.
[0026] In each of the chips for optical analysis of the
above-mentioned embodiments of the present invention, the first
substance having molecular selectivity and the second substance
having molecular selectivity are preferably antibodies or antigens,
which are of the same kinds. It is preferable that the second
substance having molecular selectivity is obtained by inactivating
the first substance having molecular selectivity, and the
inactivation is achieved by irradiation of a high energy ray
selected from an X-ray, a gamma-ray, or an electron beam, heat
treatment, electrochemical oxidation or reduction, or contact with
an acidic or alkaline buffer. Furthermore, it is preferable that
the first substance having molecular selectivity and the second
substance having molecular selectivity are DNA, and that the second
substance having molecular selectivity is obtained by replacing 10%
or less of nucleotides constituting the first substance having
molecular selectivity.
[0027] In each of the chips for optical analysis of the
above-mentioned embodiments of the present invention, two or more
observation sections each comprising the measurement region and the
reference region as one set are provided in the passage on the
substrate, and the first substances having molecular selectivity
different from each other are immobilized on the measurement region
of the observation sections, so that each observation section can
detect a different molecule.
[0028] The chip for optical analysis according to the present
invention can prepare a reference sample in which a substance to be
measured is free only by introducing a liquid sample containing the
substance to be measured into the sample introduction section, and
can measure the reference sample and a real sample containing this
substance to be measured. Furthermore, according to the present
invention, DNA, RNA, ions, amino acids, and peptides can be easily
sensed with high sensitivity.
[0029] Since the chip for optical analysis of the present invention
is provided with the means for allowing a liquid to be measured to
reach the observation section from the branching section through
the first passage earlier than reaching the observation section
from the branching section through the second passage, the
reference sample that is passed through a filter reaches the
observation section first. Therefore, even when a simple surface
plasmon resonance measurement device is used, a signal serving as
the reference of a measured value can be first obtained, and then
the liquid to be measured that is passed through the second passage
reached the observation section, resulting in an accurate analysis
of the liquid to be measured. In the chip for optical analysis of
the present invention, the observation section can be provided with
a measurement region and a reference region. With such a structure,
a reference sample can be prepared only by supplying the liquid to
be measured from the introduction port into the first passage, and
a measurement object can be detected and quantified based on an
interaction between a material having molecular selectivity
immobilized on the measurement region and the measurement
object.
[0030] According to the present invention, a change in the baseline
that occurs at the time of measurement of a real sample containing
many impurities can be minimized, and a small amount of a substance
to be measured in a sample containing many impurities can be
detected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 is a schematic diagram of a surface plasmon resonance
measurement device using the chip for optical analysis according to
the present invention;
[0032] FIG. 2A is a planar schematic diagram showing a chip for
optical analysis of one embodiment of the present invention;
[0033] FIG. 2B is a cross-sectional schematic diagram showing the
chip for optical analysis of one embodiment of the present
invention;
[0034] FIG. 3A is a planar schematic diagram showing a chip for
optical analysis of another embodiment of the present
invention;
[0035] FIG. 3B is a cross-sectional schematic diagram showing the
chip for optical analysis of another embodiment of the present
invention;
[0036] FIG. 4 (a) to (e) show manufacturing processes of the chip
for optical analysis of the present invention;
[0037] FIG. 5 (a) to (d) show manufacturing processes of the chip
for optical analysis of the present invention;
[0038] FIG. 6 shows changes over time of a substance to be measured
in a solution that reached the position of * on a passage;
[0039] FIG. 7 shows changes over time in intensity of a signal
measured at the positions of lines 1 to 3;
[0040] FIG. 8 is a cross-sectional schematic diagram showing a chip
for optical analysis of one embodiment of the present
invention;
[0041] FIG. 9 is a sagittal section view at IX-IX in FIG. 8;
[0042] FIG. 10 is a sagittal section view at X-X in FIG. 8;
[0043] FIG. 11 is an extracted and enlarged stereoscopic
three-dimensional view transparently showing a part of a
measurement region of the chip for optical analysis shown in FIG.
8;
[0044] FIG. 12 is a schematic graph showing measurement results
using the chip for optical analysis shown in FIG. 8 as changes in
current values;
[0045] FIG. 13 is an extracted and enlarged stereoscopic
three-dimensional view transparently showing a part of a second
passage in the chip for optical analysis of another embodiment of
the present invention;
[0046] FIG. 14 is a cross-sectional view showing a chip for optical
analysis of another embodiment of the present invention;
[0047] FIG. 15 is a sagittal section view at XV-XV shown in FIG.
14;
[0048] FIG. 16A is a planar schematic diagram showing a chip for
optical analysis of one embodiment of the present invention;
[0049] FIG. 16B is a cross-sectional schematic diagram showing a
chip for optical analysis of one embodiment of the present
invention;
[0050] FIG. 17 is a schematic diagram showing a chip for optical
analysis of another embodiment of the present invention;
[0051] FIG. 18 (a) to (d) are views for explaining manufacturing
processes of the chip for optical analysis shown in FIG. 16;
[0052] FIGS. 19 (a) to (d) are views for explaining other
manufacturing processes of the chip for optical analysis
corresponding to FIG. 16;
[0053] FIG. 20A is a schematic diagram showing an electrochemical
device used for inactivation of a substance having molecular
selectivity in a reference region in FIG. 19;
[0054] FIG. 20B is a schematic diagram showing the chip for optical
analysis of the present invention;
[0055] FIG. 21 is a schematic diagram of a case where a flow cell
is provided in the chip for optical analysis;
[0056] FIG. 22 is a graph showing the relationship between
resonance angles and signal intensity when a surface plasmon
resonance was measured in the measurement region and the reference
region of the chip for optical analysis;
[0057] FIG. 23 is a graph showing the relationship between time and
{.theta.(measurement region)-.theta.(reference region)} when a
surface plasmon resonance was measured in the chips for optical
analysis I, II, III, and IV; and
[0058] FIG. 24 is a graph showing the relationship between time and
{.theta.(measurement region)-.theta.(reference region)} for results
of response to an antigen and the baseline when a surface plasmon
resonance was measured in the chip for optical analysis I.
BEST MODE FOR CARRYING OUT THE INVENTION
[0059] Embodiments in which the chip for optical analysis according
to the present invention is applied to an analysis of a liquid
sample using a surface plasmon resonance measurement device will be
explained in detail with reference to the drawings. The scope of
the present invention is not limited to these embodiments, these
embodiments can be combined, and the concepts of the present
invention described in claims that fall within the scope of the
present invention can be changed or modified. In other words, it is
noted that the present invention can be naturally applied to other
arbitrary techniques attributable to the spirit thereof. For
example, the present invention can be applied not only to surface
plasmon resonance measurement devices, but also to sensing by a
total reflection type optical assay system using a material having
molecular selectivity as a probe molecule, for example, attenuated
total reflection type measurement methods using an ultraviolet
light, a visible light, or an infrared light, absorption/emission
spectrometry utilizing total reflection light excitation, and the
like.
[0060] The concept of the surface plasmon resonance measurement
device is shown in FIG. 1. The surface plasmon resonance
measurement device 1 has a light source 2 for irradiating the
measurement region of a chip for optical analysis 10 with a
measurement light Lm, a semicylindrical collecting lens
(cylindrical prism) 3 overlapped with the chip for optical analysis
10 via a matching oil, a light detector 4 for detecting a reflected
light Lr reflected on the measurement region of the chip for
optical analysis 10, and a signal processing section (not shown)
for processing a signal detected by this light detector 4.
[0061] A light from the light source 2 is linearly focused with the
collecting lens 3 and irradiated on the measurement region of the
chip for optical analysis 10. A polarizer (not shown) is placed
between the collecting lens 3 and either the light source 2 or the
detector 4 to detect only a p-polarized component. The reflected
light Lr is guided to the light detector 4 through the collecting
lens 3 again, the signal is processed at the signal processing
section (not shown), and a liquid to be measured supplied to the
chip for optical analysis 10 is analyzed. The chip for optical
analysis 10 shown in FIG. 1 comprises a substrate 11 and a chip
main body 12 joined to a surface of the substrate 11.
[0062] Hereafter, such a chip for optical analysis as described
above will be explained. The chip for optical analysis of the
present invention can be classified into the following aspects.
[0063] (1) An adsorption region (filter region) is provided between
a sample introduction section and an observation section in the
passage of the chip for optical analysis. Consequently, a
measurement object is initially adsorbed in the adsorption region,
and a reference sample reaches the observation section.
Subsequently, when the adsorption region is saturated with the
measurement object, or adsorption of the measurement object is
stopped, a sample containing the measurement object reaches the
observation section, and the measurement object can be
observed.
[0064] (2) The passage of the chip for optical analysis (main
passage) is provided with a bypass passage to generate a time lag
between samples passed through the main passage and the bypass
passage. In this aspect, it is preferable to further provide the
main passage with an adsorption region (filter region). In this
aspect, a measurement object is adsorbed in the adsorption region
in the main passage, and a reference sample reaches the observation
section. Meanwhile, a sample containing the measurement object is
allowed to flow through the bypass passage and reaches the
observation section later than the reference sample flowing through
the main passage.
[0065] (3) An observation section of the chip for optical analysis
is provided with a measurement region and a reference region. In
this aspect, a substance having molecular selectivity is
immobilized on the measurement region, and a substance which does
not have molecular selectivity and is comparable to those of the
substance having molecular selectivity immobilized on the
measurement region in other characteristics is immobilized on the
reference region. In this aspect, a sample solution containing
impurities is allowed to flow through the measurement region and
the reference region at the same time, and impurities are adsorbed
to or accumulated in the measurement region and the reference
region at the same time. As a result, when a difference between the
measurement region and the reference region is measured, effects of
adsorption and accumulation of impurities are offset, and a change
in the baseline for measurement is minimized.
[0066] In the present invention, the above-mentioned aspects (1) to
(3) can be implemented individually, or two or more thereof can be
combined.
[0067] Hereafter, the chip for optical analysis according to the
first embodiment and the second embodiment of the present invention
will be explained. The first embodiment corresponds to an aspect of
a combination of the above-described (1) and (3). The second
embodiment corresponds to an aspect of a combination of the
above-described (2) and (3) where an adsorption region is
provided.
[0068] The chip for optical analysis according to the first
embodiment is a chip for optical analysis comprising a substrate
and a passage composed of a porous membrane on the substrate,
wherein the passage comprises a sample introduction section, an
observation section, and an adsorption region for adsorbing a
substance to be measured between the sample introduction section
and the observation section, and the observation section comprises
at least a measurement region for detecting a substance to be
measured and a reference region.
[0069] The chip for optical analysis according to the second
embodiment further comprises a passage that bypasses the adsorption
region from the sample introduction section to the observation
section, so that a sample passed through the bypass passage should
reach the observation section taking more time than a sample passed
through the adsorption region.
[0070] Hereafter, the present invention will be explained with
reference to the drawings.
[0071] FIGS. 2A and 2B show the chip for optical analysis 100
according to the first embodiment of the present invention. FIG. 2A
is a planar view, and FIG. 2B is a cross-sectional view at
IIB-IIB.
[0072] The chip for optical analysis of the present invention
includes a passage 104 composed of a porous membrane on a substrate
102. A sample introduction section 106 for delivering a sample by
drops is provided at one end of this passage. The sample
introduction section is not limited so long as it has a structure
that enables delivery of a liquid sample by drops, and does not
need to be formed as a distinct region that can be distinguished
from other regions of the passage. Therefore, it is preferable to
form this section with the same material as that of the
passage.
[0073] Furthermore, the passage is provided with an observation
section 116 comprising lines 1 to 3 (114, 112, 110). The
observation section 116 comprises a reference region 118 comprising
lines 1 and 2 (114, 112) and a measurement region for detecting a
substance to be measured (110 in FIGS. 2A and 2B). FIGS. 2A and 2B
show an example of the reference region provided with two types of
lines, but a line of one type may be provided. Furthermore, the
passage is provided with an adsorption region 108 for adsorbing a
substance to be measured between the sample introduction section
and the observation section. An adsorbent immobilized on the
adsorption region may selectively adsorb the substance to be
measured or adsorb two or more substances containing the substance
to be measured.
[0074] On the measurement region, a substance having molecular
selectivity that selectively binds to a substance to be measured is
immobilized. Specific examples thereof include materials which are
antibodies, antigens, enzymes, oligonucleotides, DNA, RNA, modified
cyclodextrin, or naturally occurring or synthesized cyclic
compounds that function as ionophores. For example, when a human
IgG antigen is detected, it is sufficient to immobilize an
anti-human IqG.
[0075] A reference substance that does not bind to a substance to
be measured but is similar to a substance having molecular
selectivity is immobilized on the reference region. For example, it
is sufficient to immobilize a reference substance that causes a
different interaction between the substance having molecular
selectivity immobilized on the measurement region and the substance
to be measured. Specifically, for example, when an antibody (for
example, an anti-human IgG) is immobilized on the measurement
region, it is sufficient to immobilize an antibody of an origin or
species different from that of this antibody (for example,
anti-goat IgG) on the reference region. As shown in FIGS. 2A and
2B, when the reference region is provided with two lines, it is
preferable to immobilize such a reference substance as described
above on one of these lines and form a portion composed of a
material not containing the substance having molecular selectivity
or the reference substance such as a blocking agent (for example,
bovine serum albumin) on the other line. By doing this, detection
can be achieved with higher precision when a blocking agent is
applied to the measurement region and the region on which the
reference substance is immobilized.
[0076] When the reference region is provided with a line of one
type, a reference substance may be immobilized on the line as
described above, or a line not containing a substance having
molecular selectivity or a reference substance may be formed.
[0077] On the adsorption region, a material that can adsorb a
substance to be measured is immobilized as an adsorbent. For
example, a material that can adsorb a substance to be measured,
such as protein A, dextran or a modified product thereof, for
example, dextran gel using protein A as a modifier or modified
dextran microparticles, silica having an organic material coating,
and alumina having an organic material coating, is used. The
adsorbent may adsorb two or more substances including the substance
to be measured.
[0078] The material used for the passage is not particularly
limited so long as a liquid sample can move from the sample
introduction section to the observation section without using a
special operation such as suction, and a porous membrane can be
formed thereon. For example, cellulose or a cellulose derivative
such as cellulose acetate or nitrocellulose can be used.
[0079] The substrate is not particularly limited so long as it is
made of a material that is transparent to a light used in a
measurement device (for example, a surface plasmon resonance [SPR]
device) on which the chip for optical analysis is mounted, and any
material can be used. For example, a glass substrate, a plastic
substrate, or the like can be used.
[0080] Although not shown in FIGS. 2A and 2B, it is preferable that
the chip for optical analysis of the present invention comprises a
metal thin film between the substrate and the passage. For the
metal thin film, for example, a material such as gold can be used.
By providing a metal thin film such as a gold thin film, the chip
for optical analysis of the present invention can be used as an
analysis chip for a surface plasmon resonance device.
[0081] The operation of the chip for optical analysis of the first
embodiment will be explained. After a liquid sample containing a
substance to be measured is introduced into the chip for optical
analysis from the sample introduction section, the liquid sample is
allowed to flow along the passage in the direction of the
observation section due to capillary phenomenon of a porous
material forming the passage. When the liquid sample is allowed to
flow, first, the liquid sample reaches the adsorption region in
which an adsorbent exists. Since the adsorption region contains an
adsorbent for adsorbing the substance to be measured, the substance
to be measured is adsorbed, and other substances flow to the
observation region. Here, since the liquid sample flowing to the
observation region does not contain the substance to be measured
any longer, it means that a reference sample has reached the
observation region.
[0082] When the liquid sample is further allowed to flow, the
adsorbent of the adsorption region is saturated and does not adsorb
the substance to be measured any longer since the amount of the
adsorbent of the adsorption region is limited. In this state, the
substance to be measured reaches the observation region, and the
substance to be measured can be measured.
[0083] Thus, in the chip for optical analysis of the first
embodiment, the reference sample reaches the observation section
immediately after the introduction of the liquid sample which
includes until the time when the adsorption region is saturated
with the substance to be measured. This measurement result of the
reference sample immediately after the introduction of the liquid
sample serves as the baseline for measurement. Subsequently, after
an elapse of time, the liquid sample containing the substance to be
measured reaches the observation section, and the substance to be
measured is measured.
[0084] For the chip for optical analysis of the present invention,
a reference sample does not need to be separately prepared, and
both a signal as the baseline for measurement and a measurement
signal of a sample containing the substance to be measured can be
measured sequentially only by an operation of introducing a liquid
sample containing the substance to be measured into the chip for
optical analysis.
[0085] The chip for optical analysis 200 corresponding to the
second embodiment of the present invention will be explained with
reference to FIGS. 3A and 3B. FIG. 3A is a planar view of the chip
for optical analysis of the second embodiment, and FIG. 3B is a
cross-sectional view of FIG. 3A at IIIB-IIIB. As shown in FIGS. 3A
and 3B, the chip for optical analysis of the second embodiment
comprises a main passage 204 similar to the one explained in FIGS.
2A and 2B on a substrate 202. The main passage is provided with a
sample introduction section 206 at one end thereof for delivering a
sample by drops. Furthermore, the main passage is provided with an
observation section 208 comprising lines 1 to 3 (214, 212, 210).
The observation section 208 comprises a reference region 216
comprising lines 1 and 2 (214, 212) and a measurement region for
detecting a substance to be measured (210 in FIGS. 3A and 3B). The
main passage is further provided with an adsorption region 218 for
selectively adsorbing the substance to be measured between the
sample introduction section and the observation section. FIGS. 3A
and 3B show an example of the reference region provided with lines
of two types, but the reference region may be provided with a line
of one type. Features such as structures of the main passage, the
substrate, the adsorption region, and the observation section,
materials, and the like are as explained in the first
embodiment.
[0086] The chip for optical analysis of the second embodiment
comprises a bypass passage 220 that bypasses the adsorption region
from the sample introduction section to the observation section.
The bypass passage is composed of a porous membrane and is
positioned so that a sample passed through the bypass passage
should reach the observation section taking more time than a sample
passed through the adsorption region. So long as this condition is
satisfied, the shape, the material, and the like of the bypass
passage are not limited. For example, the bypass passage may be
rectangular as shown in FIGS. 3A and 3B or ladder-like as shown in
FIG. 8 described later. In the chip for optical analysis of the
present invention, porous membranes forming the main passage and
the bypass passage are preferably of the same material.
[0087] In the chip for optical analysis of the present invention,
the passage composed of a porous membrane has a thickness of 300 to
10,000 nm (10 .mu.m), preferably 300 to 1000 nm. Furthermore, the
full-length of the passage, the positions of the measurement region
and the reference region on the substrate, and the like can be
suitably selected to suite the optical measurement method (for
example, a surface plasmon resonance measurement method), and such
selection can be easily made by those skilled in the art.
[0088] The operation of the chip for optical analysis of the second
embodiment will be explained below. After a liquid sample
containing a substance to be measured is introduced into the chip
for optical analysis from the sample introduction section, the
liquid sample are allowed to flow in the main passage direction and
the bypass passage direction due to capillary phenomenon of the
porous material.
[0089] The liquid sample flowing in the main passage direction
first reaches the adsorption region in which an adsorbent exists.
Since an adsorbent that adsorbs the substance to be measured is
contained in the adsorption region, the substance to be measured is
adsorbed, and other substances are allowed to flow to the
observation region. Here, since the liquid sample flowing to the
observation region does not contain the substance to be measured,
it means that a reference sample reaches the observation
region.
[0090] Meanwhile, since the liquid sample flowing in the bypass
passage direction directly reaches the observation section without
passing through the adsorption region, it still contains the
substance to be measured. Furthermore, since the liquid sample
flowing via the bypass passage reaches the observation section
taking more time than the sample passed through the adsorption
region (sample flowing via the main passage), it reaches the
observation section later than the reference sample which flows via
the main passage and in which the substance to be measured is free
reaches the observation section.
[0091] Thus, the liquid sample flowing via the main passage serves
as a reference sample, and the liquid sample flowing via the bypass
passage serves as a measurement sample. Since a time lag is
generated between the liquid samples flowing via the main passage
and the bypass passage to reach the observation section, the
reference sample and the substance to be measured can be measured
by introducing a sample once.
[0092] A method for manufacturing the chip for optical analysis of
the present invention will be explained with reference to the
drawings.
[0093] First, a metal thin film 304 such as gold or the like is
formed on a substrate 302 by vapor-deposition or the like (FIG. 4
(a)). The metal thin film has a membrane thickness of about 50 nm
when used for, for example, a surface plasmon resonance (SPR)
device. Subsequently, a pattern is formed with a resist or a tape
306 on the metal thin film, so that the passage shape should be
exposed (FIG. 4 (b)). In the case of a chip for optical analysis
having such a bypass passage 220 as shown in FIGS. 3A and 3B, a
pattern including a bypass passage can be formed. Subsequently, an
organic solvent solution of a cellulose derivative such as
cellulose acetate is applied by a spin coat method or the like and
dried to remove the organic solvent, and the resist or the tape is
removed to form a passage portion 308 (FIG. 4 (c)).
[0094] Subsequently, a solution containing an adsorbent that
adsorbs a substance to be measured is applied to a portion
corresponding to the adsorption region 310 of the passage to
immobilize the adsorbent on the passage (FIG. 4 (d)). When the
adsorbent is applied, a portion corresponding to the adsorption
region may be activated, if necessary. For example, when protein A
is used as an adsorbent, it is preferable to apply an aqueous
carbodiimide solution to the adsorption region beforehand to
activate a porous material.
[0095] Subsequently, a substance having molecular selectivity and a
reference substance are immobilized on a portion corresponding to
the observation section of the passage. The substance having
molecular selectivity can be immobilized by adding dropwise a
solution containing the substance having molecular selectivity 316
on to the measurement region 314 of the observation section 312 and
drying the solution. The reference substance can be immobilized by
adding dropwise a solution containing a reference substance 320
that does not bind to the substance to be measured but is similar
to the substance having molecular selectivity onto the reference
region 318 of the observation section and drying the solution (FIG.
4 (e)). When the reference region is provided with two lines 322,
324 as shown in FIGS. 2A and 2B, a reference region neither
containing a substance having molecular selectivity nor a reference
substance may be formed by immobilizing the reference substance on
one of these lines as described above and adding dropwise a
solution containing a blocking agent 326 onto the other and drying
the solution. When the blocking agent is added dropwise onto the
measurement region and the reference region containing the
reference substance to block these regions, it is preferable to
provide a region containing a blocking agent, so that the
measurement conditions should be uniform.
[0096] Another method for manufacturing the chip for optical
analysis of the present invention will be explained.
[0097] As described above, a metal thin film 304 is formed on a
substrate 302, and a pattern is formed on the metal thin film with
a resist or a tape 306, which pattern has the passage shape that
exposes the metal thin film. At this time, a portion corresponding
to the adsorption region 310 of the passage is not exposed the
metal thin film (FIG. 5 (a)). Furthermore, when such a bypass
passage 220 as shown in FIG. 3 is formed, a pattern including a
bypass passage can be formed. Subsequently, an organic solvent
solution of a cellulose derivative such as cellulose acetate is
applied by a spin coat method or the like and dried to remove the
organic solvent, and the resist or the tape is removed to form a
passage portion 402 separated by the adsorption region (FIG. 5
(b)).
[0098] Subsequently, a solution containing an adsorbent that
adsorbs a substance to be measured (preferably, a solution mixed
with a porous material for forming the passage) is applied to the
adsorption region 310, the solvent or the like is removed, and an
adsorption region is formed so as to connect to the passage portion
402 (FIG. 5 (c)). Subsequently, the observation section is formed
as described above, and thus a chip for optical analysis in which
the passage 308 is formed can be manufactured (FIG. 5 (d)).
[0099] In the present specification, "immobilization" encompasses
chemical or physical binding onto the passage including the case
where an adsorbent, a substance having molecular selectivity, a
reference substance, and the like are adsorbed to the passage as
described above.
[0100] In the above explanation of the first and second
embodiments, an example where a porous membrane is used for the
passage, and the sample moves by capillary phenomenon has been
shown, but the present invention is not limited to this example.
For example, sucking-up means such as a pump can be provided at a
portion downstream of the passage without using a porous membrane
for the passage. Furthermore, the above-mentioned aspects (1) and
(3) of the present invention are combined in the above-described
first embodiment, and the above-mentioned aspects (2) (an
adsorption region is provided) and (3) of the present invention are
combined in the second embodiment, but the present invention is not
limited to these embodiments. A chip for optical analysis can be
constituted by the above-mentioned aspect (1) of the present
invention solely, or the above-mentioned aspect (2) of the present
invention solely. In these cases, the chip can be constituted so
that a target substance can be suitably measured at the observation
section.
[0101] A chip for optical analysis 10 of another embodiment (third
embodiment) used for the surface plasmon resonance measurement
device 1 will be explained. This chip for optical analysis
corresponds to a combination of the above-mentioned aspects (2) (an
adsorption region is provided) and (3) of the present
invention.
[0102] The cross-sectional structure of the chip for optical
analysis 10 is shown in FIG. 8. The sagittal section structure
thereof at IX-IX and the sagittal section structure thereof at X-X
are shown in FIGS. 9 and 10, respectively. The chip for optical
analysis 10 in this embodiment has a substrate 11 and a chip main
body 12 joined to a surface of the substrate 11. The substrate 11
in this embodiment is a thin plate having, for example, a length of
10 mm, a width of 16 mm, and a height of 1 mm and is made of glass
or plastic transparent to a measurement light Lm. Furthermore, the
chip main body 12 in this embodiment is a rectangular solid shape
having, for example, a length of 7 mm, a width of 10 mm, and a
height of 4 mm, and a resin that can be easily machined, etched, or
the like is used. Polydimethylsiloxane (PDMS) processed into a
predetermined shape is used in this embodiment.
[0103] A gold thin film layer 14 (see FIG. 11) with a rectangular
pattern having a thickness of about 50 nm facing a first passage 13
positioned at the observation section Zm surrounded by a two-dot
chain line in FIG. 8 when the chip main body 12 is joined can be
formed on a surface of the substrate 11 by vapor-deposition or the
like. It is preferable to provide the observation section Zm
virtually at the center of the substrate 11. An appearance of this
gold thin film layer 14 portion is enlarged and shown in FIG. 11.
An antibody as a material having molecular selectivity is
immobilized on the observation section on the gold thin film layer
14 to form antibody-immobilized regions A, B, and C. In this
embodiment, three antibody-immobilized regions A, B, and C can be
formed so as to cross the first passage 13 by limiting the amount
of a liquid containing an antibody added dropwise onto the gold
thin film layer 14. These three antibody-immobilized regions A, B,
and C in this embodiment can contain anti-human IgG, anti-rabbit
IgG, and anti-goat IgG, which have selectivity to different
antigens. These antibody-immobilized regions A, B, and C can be
formed by adding dropwise liquids containing the corresponding
antibodies onto the predetermined antibody-immobilized regions A,
B, and C on the gold thin film layer 14 and allowing to stand for a
predetermined time, then sucking the remaining liquids, and
attaching a part of antibodies contained in the liquids onto a
surface of the gold thin film layer 14.
[0104] Meanwhile, a through hole 15 of about 0.6 mm serving as a
discharge port is formed at the center in the width direction
(vertical direction in FIG. 8) on one end side in the longitudinal
direction of the chip main body 12 (horizontal direction in FIG.
8). Furthermore, a stepped hole 16 forming a part of the first
passage 13 is formed at the center in the width direction on the
other end side in the longitudinal direction of the chip main body
12. The inside diameter of a small-diameter portion 16a of the
stepped hole 16 serving as an introduction port is, for example,
about 0.45 mm, and the Inside diameter of a large-diameter portion
16b filled with a filter 17 described later is, for example, 0.6
mm. Furthermore, a blind hole 19 having an inside diameter of, for
example, 2.5 mm serving as a liquid collecting section of the
second passage 18 is formed adjacent to this stepped hole 16 at one
corner on the other end side in the longitudinal direction of the
chip main body 12. The bottom part of this blind hole 19 and the
large-diameter portion 16b of the stepped hole 16 are communicated
with each other via a continuous hole 20 which is a part of the
second passage 18. Specifically, the connection section of the
continuous hole 20 and the large-diameter portion 16b of the
stepped hole 16 serves as a branching section in the present
invention. In this embodiment, since the diameter of the blind hole
19 is set very large as compared with the diameter and the length
of the continuous hole 20, the continuous hole 20 can be formed by
inserting into the blind hole 19 a drill head for drilling the
continuous hole 20 from this blind hole 19. Formation methods other
than this method can be used, but the number of processing
processes may be increased, or an additional process for preventing
a liquid spill may be required. A groove 22 in a straight line
having a width of, for example, 0.5 mm and a depth, that is, a
height of, for example, 50 .mu.m that is communicated with the
through hole 15 and the large-diameter portion 16b of the stepped
hole 16 as a part of the first passage 13, and a ladder-like groove
23 having, for example, a width of 0.3 mm and a height of 50 .mu.m
that is communicated with the halfway of this groove 22 on one end
side thereof and communicate with the blind hole 19 as a part of
the second passage 18 on the other end side are carved in the joint
surface 21 with the substrate 11 of this chip main body 12. The
connection section with the groove 22 that is in a straight line
with an end of this ladder-like groove 23 is a merging section of
the two passages 13, 18, and the first passage 13 positioned at the
center in the longitudinal direction of the groove 22 in a straight
line downstream thereof serves as an observation section Zm. A
spiral or maze-like groove can be formed instead of the ladder-like
groove 23, and these shapes, cross-sectional structures, and the
like are basically arbitrarily selected.
[0105] A filter 17 for capturing a predetermined component
contained in a liquid to be measured (also referred to as an
adsorbent in the present specification) is filled on the downstream
side where the side is the connection section of the stepped hole
16 with the continuous hole 20 in the large-diameter portion 16b of
the stepped hole 16 in the first passage 13. As the filter 17 in
this embodiment, gel-like microparticles using active carbon,
molecular sieve, dextran, cellulose, agarose, or curdlan as the
main material, silica modified with an organic matter containing a
hydrophobic group, alumina microparticles, or the like can be used.
This filter 17 is held by a porous holding member 24 made of glass
fiber injected into the large-diameter portion 16b of the stepped
hole 16 at the central portion of the large-diameter portion 16b of
the stepped hole 16, so that the inlet portion of the continuous
hole 20 should not be blocked.
[0106] Plasma treatment is performed on the joint surface 21 of the
chip main body 12 formed as described above, the joint surface 21
of the chip main body 12 and the surface of the substrate 11 are
overlapped and pressed to each other, so that a gold thin film
layer 14 formed on the surface of the substrate 11 should be
positioned at a predetermined position of the groove 22 in a
straight line, that is, the observation section Zm, and the joint
surface 21 of the chip main body 12 is joined to the surface of the
substrate 11 in an integrated fashion. Thus, the first passage 13
reaching the groove 22 that forms a straight line from the
small-diameter portion 16a of the stepped hole 16 via the
large-diameter portion 16b and the second passage 18 reaching the
ladder-like groove 23 from the continuous hole 20 through the blind
hole 19 are formed to prepare a chip for optical analysis 10. It is
noted that, so long as the volume of the first passage 13 is
sufficiently smaller than that of the second passage 18, these
passages 13, 18 may have any form, which is not limited to the
embodiments described above.
[0107] When measurement is performed using the surface plasmon
resonance measurement device shown in FIG. 1, a tube for supplying
a liquid to be measured (not shown) is connected to the
introduction port of the chip for optical analysis 10, specifically
the small-diameter portion 16a of the stepped hole 16, and a
phosphate buffer having a concentration of 0.1 mol/L containing
antigens which are measurement objects, specifically human IgG
(sample a) and rabbit IgG (sample b), and albumin as a liquid to be
measured, which is an impurity, is supplied into the chip for
optical analysis 10 using a micro syringe pump (not shown). Then,
the liquid that has been measured and passed through the
observation section Zm is discharged out of the chip for optical
analysis 10 using a discharge port, specifically a waste fluid tube
(not shown) connected to the through hole 15. Meanwhile, a
measurement light from the light source 2 is irradiated on the
observation section Zm from the substrate 11 of the chip for
optical analysis 10, a reflected light thereofr guided to the light
detector 4 is processed at the signal processing section, and the
refractive index and changes in the refractive index are obtained.
These measurement results are shown in FIG. 12.
[0108] The liquid to be measured is passed through the filter 17
from the first passage 13. Meanwhile, the antigens and albumin,
which is an impurity, are adsorbed to the filter 17 so as to be
used as a reference sample, and the liquid reaches the observation
section Zm of the first passage 13 at the time of t.sub.1. A signal
of the surface plasmon resonance angle detected here, specifically
a refractive Index N serves as the measurement reference.
[0109] Subsequently, the liquid to be measured containing the
impurity and the antigens from the second passage 18 is allowed to
flow from the merging section of the first passage 13 into the
first passage 13 and reaches the observation section Zm at the time
of t.sub.2, which is later than t.sub.1, and the mixing ratio
thereof becomes stable after t.sub.3. Here, the impurity and the
antigens which are the measurement objects in the liquid to be
measured are adsorbed to the antibody-immobilized regions A, B, and
C of the observation section. Albumin as an impurity is equally
adsorbed to all the three antibody-immobilized regions A, B, and C,
but human IgG and rabbit IgG, antigens, are selectively adsorbed to
the corresponding antibody-immobilized regions A and B,
respectively. As a result, differences between the
antibody-immobilized region C, on which anti-goat IgG is
immobilized, and other two antibody-immobilized regions A and B are
detected as changes in the refractive indices a and b corresponding
to signals by human IgG and rabbit IgG. In an antigen-antibody
reaction in a surface modification format using a total
reflection-type optical system, the reaction rate is proportional
to the antigen concentration, and human IgG in the liquid to be
measured can be quantified from changes over time of the surface
plasmon resonance angle. That is, human IgG and rabbit IgG can be
quantified from changes over time in the surface plasmon resonance
angles represented as changes in the refractive indices a and
b.
[0110] In the case of a liquid to be measured containing many
impurities that are hardly passed through the filter 17
incorporated in the first passage 13 or a liquid to be measured
with high viscosity, a greater difference needs to be generated in
time t.sub.1, t.sub.2 to reach the observation section Zm from the
branching section through the first and second passages 13, 18. To
this end, a hydrophilically treated layer may be formed on the wall
surface of the second passage 18. For example, a silane coupling
agent having an amino group can be applied on the inner peripheral
wall of the ladder-like groove 23 of the above-described
embodiments and the surface of the substrate 11 forming the second
passage 18 together therewith to form a hydrophilically treated
layer. Since a hydrophilic silane coupling material has an action
of drawing the water content in a phosphate buffer in the second
passage 18 in contact therewith, the flow of the phosphate buffer
in the second passage 18 is suppressed, time t.sub.2 to reach the
observation section Zm is further increased.
[0111] Similarly, as means for generating a great difference in
time t.sub.1, t.sub.2 to reach the observation section Zm from the
branching section through the first and second passages 13, 18, it
is also effective to form one or more passage resistance-increasing
blocks 25 protruded from the surface of the substrate 11 facing the
second passage 18 into the second passage 18, as shown in FIG. 13,
at a predetermined interval or randomly. The passage
resistance-increasing block 25 in this embodiment constitutes a
cylinder having, for example, a diameter of 50 .mu.m and a height
of 30 .mu.m and is formed as a pattern on the surface of the
substrate 11 using a resist. Thus, the resistance of the second
passage 18 can be increased by allowing the passage
resistance-increasing block 25 to exist in the second passage 18,
and time to reach the observation section Zm can be increased.
[0112] In the above-described third embodiment, the liquid that has
been measured is discharged out of the chip for optical analysis 10
from the discharge port, specifically the through hole 15, but it
can be held in the chip for optical analysis 10.
[0113] An example of the chip for optical analysis 10 in such a
modified form is shown in FIGS. 14 and 15. FIG. 14 shows a
cross-sectional structure, and FIG. 15 shows sagittal section
structure at XV-XV in FIG. 14. In these figures, components having
the same function as in the above-described embodiments are only
labeled with the same symbols, and the same explanation will be
omitted. In this embodiment, instead of the above-described through
hole 15, a void portion 27 housing an absorption body 26 is formed
in the chip main body 12 so as to connect to an end of the first
passage 13. An absorption body 26 such as a pulp or a gelated
polymer is housed for absorbing and holding a liquid in this void
portion 27. In this embodiment, since the absorption body 26
absorbs and holds the measured fluid when the measured fluid
reaches the absorption body 26 in the void portion 27 from the
first passage 13, the measured fluid introduced into the chip for
optical analysis 10 from an introduction port, specifically the
small-diameter portion 16a of the stepped hole 16, can be
automatically sucked, and the measured fluid can also be led from
the second passage 18 to the absorption body 26 without operating a
measured fluid supplying pump.
[0114] In the chip for optical analysis of the third embodiment,
the passage may be a narrow tube, but otherwise the passage can be
formed with a porous material. Examples of the porous material
include those explained in the first and second embodiments.
Furthermore, the third embodiment shows an example of a combination
of the above-mentioned aspects (2) (adsorption region is provided)
and (3) of the present invention, but the scope of the present
invention is not limited to this example. The chip for optical
analysis can be constituted by the above-mentioned aspect (2) of
the present invention solely. In this case, it is sufficient to
constitute the chip for optical analysis, so that a target
substance can be suitably measured at the observation section.
[0115] Since the chip for optical analysis according to the
above-described third embodiment is provided with means for
allowing a liquid to be measured to reach the observation section
from the branching section through the first passage earlier than
reaching the observation section from the branching section through
the second passage, it has the following effects in addition to the
above-described effects.
[0116] When there is provided a liquid absorption holding section
which is incorporated in the chip main body and is connected to the
first passage on the downstream side of the observation section for
absorbing a liquid to be measured passed through the observation
section, an electric-powered pump for allowing the liquid to be
measured to flow into the chip for optical analysis or the like can
be omitted. Furthermore, since the waste fluid after measurement is
held in the liquid absorption holding section and is not allowed to
flow out, post-treatment becomes easy.
[0117] When a hydrophilically treated layer is formed on the wall
surface of the second passage, the flow of a liquid to be measured
in the second passage is suppressed since the water component in
the liquid to be measured passed through the second passage has
affinity with the hydrophilically treated layer. As a result, the
liquid to be measured can reach the observation section from the
branching section through the first passage earlier than reaching
the observation section from the branching section through the
second passage.
[0118] When at least one passage resistance-increasing block
protruded into the second passage from the surface of the substrate
facing the second passage is formed, the passage
resistance-increasing block suppresses the flow of the liquid to be
measured in the second passage. As a result, the liquid to be
measured can reach the observation section from the branching
section through the first passage earlier than reaching the
observation section from the branching section through the second
passage.
[0119] When the volume of the first passage reaching the merging
section from the branching section is set larger than that of the
second passage, the liquid to be measured can reach the observation
section from the branching section through the first passage
earlier than reaching the observation section from the branching
section through the second passage. In particular, when a liquid
collecting section for collecting the liquid to be measured is
positioned in the middle of the second passage, a reference sample
reaching the observation section through the filter and a fluid to
be measured reaching the observation section through the second
passage can reach the observation section with a great time
lag.
[0120] When a material having molecular selectivity such as an
antibody or an antigen is immobilized on the observation section of
the first passage, an immune reaction related to an organism can be
measured. In particular, when the material having molecular
selectivity is an enzyme, detection of a biologically-relevant
substance or a drug, quality control of food, or detection of an
environmentally acting substance is enabled. In the case of an
oligonucleoside or a ribonucleoside, information of a substance to
be measured at a gene level can be identified. In the case of a
modified cyclodextrin compound, an ion or an amino acid can be
selectively measured. Furthermore, when the filter contains a
dextran gel using protein A as a modifier, a reference sample in
which an IgG-type immunity substance is free can be prepared in
this chip for optical analysis, and a chip for optical analysis for
measuring an IgG-type immunity substance can be obtained. When at
least one of organic membrane-coated silica and organic
membrane-coated alumina is contained, a reference sample which
proteins other than IgG, amino acids, and ions are free can be
prepared in the chip for optical analysis, and a chip for optical
analysis for detecting proteins other than IgG, amino acids, and
ions can be obtained. When a measurement object contains various
kinds of impurities, the chip for optical analysis can be used by
filling a dextran gel using protein A as a modifier at the bottom
and organic membrane-coated silica or organic membrane-coated
alumina at the top.
[0121] The fourth and fifth embodiments of the present invention
will be explained. The fourth and fifth embodiments correspond to
the above-mentioned aspect (3) of the present invention.
[0122] The chip for optical analysis of the fourth embodiment
comprises a substrate, a metal thin film on the substrate, and an
observation section on the metal thin film, wherein the observation
section is provided with a measurement region and a reference
region. In the chip for optical analysis, on the measurement
region, a first substance having molecular selectivity that
selectively interacts with a specific molecule is immobilized, and
on the reference region, a second substance having molecular
selectivity that is different only in selectivity to the specific
molecule with which the first substance having molecular
selectivity interacts and is comparable to the first substance
having molecular selectivity in other characteristics is
immobilized, and preferably, the first substance having molecular
selectivity and the second substance having molecular selectivity
are immobilized on the substrate by the same method.
[0123] The chip for optical analysis of the fourth embodiment of
the present invention will be explained with reference to the
drawings.
[0124] The chip for optical analysis of the fourth embodiment of
the present invention is as shown in FIGS. 16A and 16B. FIG. 16B is
a cross-sectional view at XVIB-XVIB of FIG. 16A. This chip for
optical analysis has a metal thin film 1002, preferably a gold thin
film, formed on a substrate 1001 by a method such as
vapor-deposition, and a measurement region 1003 and a reference
region 1004 are provided on this gold thin film. The measurement
region 1003 and the reference region 1004 are preferably arranged
in parallel in a straight line as shown in the figure.
[0125] The measurement region and the reference region are arranged
along a passage of a flow cell for transporting a sample as
described later, but either the measurement region or the reference
region may be arranged closer to the inlet side of the flow
cell.
[0126] Examples of the substances having molecular selectivity used
in the measurement region and the reference region include
biologically-relevant substances such as antibodies, antigens,
enzymes, DNA, RNA, and proteins, nonbiologically-relevant
substances such as ionophores and modified products of dextran, and
so forth. Furthermore, antibodies obtained by protein engineering
that have different selectivity and similar three-dimensional
structures and the like can also be used for both regions.
[0127] It is preferable to design the substances having molecular
selectivity used in the measurement region and the reference
region, so that they should be different from a substance to be
measured only in selectivity. Such a design includes synthesis of
the substance having molecular selectivity so as to be different
from a substance to be measured only in selectivity (any chemical
or biological means) and inactivation of the substance having
molecular selectivity so as to be different from a substance to be
measured only in selectivity.
[0128] Specifically, a first substance having molecular selectivity
that selectively interacts with a substance to be measured in the
chip for optical analysis is immobilized on the measurement region.
The first substance having molecular selectivity can selectively
interact with the substance to be measured, and specific examples
thereof include such biologically-relevant substances and
nonbiologically-relevant substances mentioned above, those obtained
by protein engineering techniques, and so forth.
[0129] A second substance having molecular selectivity is
immobilized on the reference region. The second substance having
molecular selectivity is not particularly limited so long as it is
different only in selectivity to a specific molecule with which the
first substance having molecular selectivity interacts and is
comparable to the first substance having molecular selectivity in
other characteristics. That is, the second substance having
molecular selectivity is a substance that is comparable to the
first substance having molecular selectivity in characteristics,
except that it does not interact with molecules other than the
substance to be measured. In the present invention, it is
preferable to use a substance of the same type or kind as that of
the first substance having molecular selectivity as the second
substance having molecular selectivity.
[0130] As the second substance having molecular selectivity, for
example, a substance obtained by inactivating the first substance
having molecular selectivity can be used. Here, "inactivation"
means subjecting the first substance having molecular selectivity
to a specific processing, so that only selectivity to a substance
to be measured should be different from that of the first substance
having molecular selectivity. Examples of such an inactivation
processing include irradiation with a high energy ray such as an
X-ray, a gamma(.gamma.)-ray, or an electron beam, heat treatment,
electrochemical oxidation or reduction, contact with an acidic or
alkaline buffer, and so forth.
[0131] Not only the second substance having molecular selectivity
can be obtained by the above-described methods, but also when the
first substance having molecular selectivity is an antibody, an
anti-antibody of the same kinds can be used as the second substance
having molecular selectivity. For example, the case where antibody
C having selectivity to a substance to be measured c is immobilized
on the measurement region is illustrated. At this time, an
anti-antibody (antibody C') prepared by administering a substance
different from the substance to be measured c to the same animal
from which the antibody C has been obtained as an antigen (for
example, a bovine, a goat, a rabbit, a mouse, a rat, a sheep, a
dog, a feline, an equine, camel, etc.) is immobilized on the
reference region. When antibody C is IgG, IgA, IgM, IgE, IgD, or
the like, antibody C' is anti-IgG, anti-IgA, anti-IgM, anti-IgE,
anti-IgD, or the like, respectively. Whether these anti-antibodies
bind to Fc or Fab of these anti-antibodies is the same, and these
anti-antibodies are different only in selectivity to the substance
to be measured.
[0132] Furthermore, considering the case where antigen D having
selectivity to a substance to be measured d is used as the first
substance having molecular selectivity (that is, antigen D is
immobilized on the measurement region), antigen D', which is
different from the antigen D only in selectivity to the substance
to be measured d, can be used as the second substance having
molecular selectivity to be immobilized on the reference
region.
[0133] Furthermore, DNA, RNA, a protein, or the like can be used as
the first substance having molecular selectivity. These are not
particularly limited so long as they can achieve a selective
coupling with the substance to be measured, such as formation of a
conjugate.
[0134] When DNA or RNA is used as the first substance having
molecular selectivity, the second substance having molecular
selectivity can be obtained by adding, replacing, deleting, or the
like several nucleotides constituting the first substance having
molecular selectivity. Specifically, when DNA is used as the first
substance having molecular selectivity, it is preferable to obtain
the second substance having molecular selectivity by replacing 10%
or less of nucleotides constituting the first substance having
molecular selectivity.
[0135] The substrate is not particularly limited so long as it is
made of a material that is transparent to a light used in a
measurement device on which the chip for optical analysis is
mounted (for example, a surface plasmon resonance [SPR] device),
and any material can be used. For example, a glass substrate, a
plastic substrate, or the like can be used.
[0136] When a biologically-relevant substance such as an antibody
is used in the measurement region in the present invention, effects
on the reaction rate itself of an interaction between the
biologically-relevant substance and the substance to be measured,
for example, an antigen-antibody reaction are minimal even when
many impurities exist in the sample solution, since selectivity of
a biologically-relevant substance is very high. Therefore, even
when many impurities exist in the sample solution, the substance to
be measured can be detected and/or quantified with high sensitivity
and high precision.
[0137] In the present specification, interaction means a chemical
or physical action of a substance having molecular selectivity with
a substance to be measured, and examples thereof include a hydrogen
bond, an ionic bond, an intermolecular bond, uptake of a substance
into a molecule, formation of a conjugate, adsorption, and so
forth. For example, when the above-mentioned biologically-relevant
substance is used, an antigen-antibody reaction, an
enzyme-substrate interaction, a base pair bonding in formation of a
double strand of DNA or RNA, and so forth can be mentioned.
[0138] The fifth embodiment of the present invention will be
explained. The chip for optical analysis of the fifth embodiment is
as shown in FIG. 17. This chip for optical analysis has a metal
thin film 1002, preferably a gold thin film, formed on a substrate
1001 by a method such as vapor-deposition. A plurality of
measurement regions 1013, 1015 and reference regions 1014, 1016 are
provided in parallel on this gold thin film. In this embodiment,
the measurement region 1013 and the reference region 1015 and the
measurement region 1014 and the reference region 1016 as sets
constitute observation sections 1010 and 1011. In the observation
section 1010 and the observation section 1011, first substances
having molecular selectivity of different types are immobilized on
the respective measurement regions, so that different types of
molecules can be detected. Furthermore, the second substance having
molecular selectivity is immobilized in accordance with the first
substance having molecular selectivity. The first substance having
molecular selectivity and the second substance having molecular
selectivity are as described above. This embodiment shows an
example where two observation sections are provided, but the
present invention is not limited to this example, and even more
observation sections may be provided.
[0139] When a plurality of observation sections are provided on the
substrate, it is preferable to arrange each observation section
along a passage of a flow cell for transporting a sample,
preferably, so that observation sections are aligned in parallel in
a straight line.
[0140] Positions of the measurement region and the reference region
on the substrate and the like in the chip for optical analysis of
the present invention can be suitably selected to suit the optical
measurement method (for example, a surface plasmon resonance
measurement method). Such a selection can be suitably made by those
skilled in the art.
[0141] As described above, in the chip for optical analysis of the
present invention, effects of adsorption or accumulation of
impurities is offset by applying the first substance having
molecular selectivity to the measurement region and the second
substance having molecular selectivity different from the first
substance having molecular selectivity only in selectivity to the
substance to be measured to the reference region, and a change in
the baseline for measurement can be minimized when a sample is
measured.
[0142] A method for manufacturing the chips fox optical analysis of
the fourth embodiment and the fifth embodiment will be explained
with reference to the drawings.
[0143] The chip for optical analysis of the fourth embodiment can
be prepared by, for example, the procedures shown in FIGS. 18 (a)
to (d). First, a metal thin film 1002 of gold or the like is formed
on a substrate 1001 by vapor-deposition or the like (FIG. 18 (a)).
The metal thin film has a membrane thickness of about 50 nm, for
example, when used for an SPR device. Subsequently, a cover seal
1034 having openings 1030, 1032 corresponding to the measurement
region 1003 and the reference region 1004 are attached on the metal
thin film (FIG. 18 (b) (i) and (ii)). Solutions 1021, 1022 each
containing the first substance having molecular selectivity or the
second substance having molecular selectivity (for example, a
phosphate buffer solution of an antibody or an anti-antibody) are
added dropwise into these openings, respectively, and allowed to
stand for a predetermined time (FIG. 18 (c)). Then, the solutions
are removed, and the cover seal is peeled off to obtain a chip for
optical analysis in which the first substance having molecular
selectivity and the second substance having molecular selectivity
are immobilized on the measurement region and the reference region
(FIG. 18 (d)), respectively. The predetermined time for the
standing varies depending on the immobilized material, but, for
example, is several minutes to 60 min, preferably about 20 min when
a phosphate buffer solution of an antibody is used. In the present
invention, "immobilization" encompasses a chemical or physical
binding with the top of a substrate or a metal thin film including
the placement of the first substance having molecular selectivity
and the second substance having molecular selectivity on the
substrate, preferably a metal thin film as described above.
[0144] The chip for optical analysis of the fifth embodiment can be
prepared by using a cover seal provided with openings corresponding
to a plurality of observation sections shown in FIG. 18 (b) and
immobilizing a substance having molecular selectivity depending on
the substance to be measured on each observation section.
[0145] Another method for manufacturing a chip for optical analysis
having one observation section will be explained with reference to
FIGS. 19 and 20. First, a substrate 1001 is covered with a mask
1040. This mask has three openings opened in portions forming a
metal thin film 1048, 1049, 1050, openings corresponding to pad
sections 1044, 1045, and openings corresponding to joining sections
1046, 1047 connecting the metal thin film 1049 and the pad section
1045 and the metal thin film 1050 and the pad section 1044. Metal
thin films 1048, 1049, 1050 made of a material such as gold (FIG.
19 (a)) is formed through these openings by means such as
vapor-deposition.
[0146] Subsequently, the mask is removed, a cover seal 1054 having
openings 1051, 1052 is attached on portions of the metal thin films
1048, 1049, and a solution of the first substance having molecular
selectivity 1055 is added dropwise into these openings 1051, 1052
and left stand for a predetermined time (FIG. 19 (b)). Then, the
solution is removed, and the cover seal is peeled off to obtain a
substrate in which the first substance having molecular selectivity
1055 is immobilized on the metal thin films 1048, 1049 (FIG. 19
(c)).
[0147] Subsequently, as shown in FIG. 19 (d), a cover seal 1056
having a pattern of an opening 1053 with which a part of the
joining sections 1046, 1047 is covered and portions of the metal
thin films 1049, 1050 are well-shaped is attached. This well-shaped
opening 1053 can hold an electrolyte solution. An electrolyte
solution (for example, a phosphate buffer solution) is added
dropwise into the above-mentioned well 1053 of the substrate thus
obtained, and the first substance having molecular selectivity on
the metal thin film 1049 is inactivated using a silver-silver
chloride reference electrode, a potentiostat, and a function
generator as shown in FIG. 20A and used as the second substance
having molecular selectivity 1058. Finally, the reference
electrode, the electrolyte solution, the cover seal, the
potentiostat, the function generator, and the like are removed to
obtain the chip for optical analysis (FIG. 20B).
[0148] In the method for manufacturing the chip for optical
analysis of the present invention, when the second substance having
molecular selectivity is prepared by inactivating the first
substance having molecular selectivity, the first substance having
molecular selectivity may be inactivated after the first substance
having molecular selectivity is immobilized on the reference region
beforehand. Alternatively, the first substance having molecular
selectivity is inactivated beforehand, and then the inactivated
first substance having molecular selectivity may be immobilized on
the reference region.
[0149] The chip for optical analysis thus obtained is mounted on,
for example, a surface plasmon resonance device (for example, the
device described in Patent Document 1), is equipped with a flow
cell made of polydimethylsiloxane (PDMS) at the top as shown in
FIG. 21, and then is used. A liquid sample can be introduced into
the flow cell from the micro syringe pump via a tube.
[0150] When a liquid sample is introduced, a sample solution
containing impurities flows into the measurement region and the
reference region at the same time, and impurities are adsorbed or
accumulated in the measurement region and the reference region at
the same time. As a result, when a difference between the
measurement region and the reference region is measured, effects of
adsorption and accumulation of impurities are offset, and a change
in the baseline for measurement can be minimized. Therefore, since
a change in the baseline can be minimized as compared with a signal
obtained upon detection of a substance to be measured at a low
concentration in the liquid sample, a substance to be measured can
be detected or quantified with high sensitivity and high
precision.
[0151] The fourth and fifth embodiments correspond to the
above-mentioned aspect (3) of the present invention. However, the
present invention is not limited to these embodiments, and the
above-mentioned aspect (1) and/or (2) and the above-mentioned
aspect (3) can be used in combination.
[0152] The chip for optical analysis of the present invention can
be applied to total reflection optical systems and assay systems
used by immobilizing a substance having molecular selectivity and
can be applied as a chip for measurement of absorption based on not
only the surface plasmon resonance measurement methods but also
total reflection measurement methods utilizing a prism or a
diffraction grating.
EXAMPLES
[0153] The present invention will be explained more specifically
with reference to the following examples.
Example 1
[0154] This example shows a chip for optical analysis for a passage
without a bypass passage as shown in FIGS. 2A and 2B, for which a
protein A adsorbent is used as an adsorbent.
[0155] Cellulose acetate was dissolved in an organic solvent to
prepare a cellulose acetate solution. A gold thin film (thickness
50 nm) was formed on a BK7 glass substrate by vapor-deposition. A
resist membrane or a tape was applied onto this gold thin film, and
a pattern in which a portion of the gold thin film corresponding to
a passage was exposed was formed. The patterned gold/glass
substrate was mounted on a spin coater, the cellulose acetate
solution prepared beforehand was added dropwise to the substrate,
then the substrate was rotated, and the organic solvent was
evaporated to form a white membrane-form passage. Subsequently, a
buffer solution of protein A was prepared and added dropwise to an
adsorption region of the passage, and protein A was adsorbed to the
passage to form the adsorption region. The region to which protein
A was adsorbed had been activated beforehand by adding dropwise an
aqueous solution of carbodiimide and drying the solution. Thus, a
passage having a length of 10 mm, a width of 1 mm, and a thickness
of 1 .mu.m was formed.
[0156] Subsequently, a small amount of a phosphate buffer solution
containing 100 .mu.gmL.sup.-1 anti-human IgG (0.1 M, pH 6.8) was
added dropwise onto a portion of the passage corresponding to a
measurement region (a portion corresponding to line 3 in FIGS. 2A
and 2B) and dried to immobilize the antibody. Similarly, a
phosphate buffer solution containing 100 .mu.gmL.sup.-1 anti-goat
IgG was added dropwise onto a portion adjacent to the region on
which anti-human IgG was immobilized (a portion corresponding to
line 2 in FIGS. 2A and 2B) and dried to immobilize a reference
antibody.
[0157] Subsequently, a phosphate buffer solution containing 4
mgmL.sup.-1 bovine serum albumin was prepared as a blocking agent
solution, and this solution was added dropwise onto the measurement
region and the reference region on which the antibodies were
immobilized (lines 3 and 2 in FIGS. 2A and 2B) and further another
region in the reference region (a portion corresponding to line 1
in FIGS. 2A and 2B) and dried.
[0158] The prepared chip for optical analysis was mounted on a
prism of a surface plasmon resonance measurement device via a
matching oil. A phosphate buffer solution containing human IgG was
added dropwise onto a sample introduction section of the passage of
the chip for optical analysis as a liquid sample. Human IgG that is
a substance to be measured in the liquid sample is adsorbed to
protein A in the adsorption region, the phosphate buffer solution
not containing human IgG reaches to the observation region, and SPR
is observed. FIG. 6 shows profiles of the concentration of human
IgG in the liquid sample which reaches the portion represented by *
in FIG. 2B. As shown in FIG. 6, the reference sample not containing
human IgG reaches the measurement region at the time of t0. The
angle of SPR observed from time t0 to time t1, at which the liquid
sample containing human IgG reaches the measurement region, serves
as a base level for measurement.
[0159] Subsequently, after the adsorbent region is saturated by the
liquid sample is further added dropwise, or substance to be
measured is prevented from being adsorbed to the adsorbent by
protecting (or covered) the adsorption region with a plastic or the
like, a large volume of the liquid sample is introduced through the
sample introduction section, and thus the liquid sample containing
human IgG reaches the measurement region, and human IgG binds
selectively to the anti-human IgG in the measurement region. On the
other hand, neither of the region on which the anti-goat IgG was
immobilized and the region on which only a blocking agent was
immobilized in the reference region binds to human IgG. Therefore,
at this stage, the angle of SPR increases only in the measurement
region (a portion corresponding to line 3 in FIGS. 2A and 2B), and
the angle of SPR only slightly increases in the reference region
(lines 2 and 1 in FIGS. 2A and 2B) as shown in FIG. 7. The
difference between the measurement region (line 3) and the region
on which anti-goat IgG was immobilized (line 2) in the reference
region represents selective binding of human IgG to anti-human IgG,
and human IgG can be quantified from these changes over time in the
increase in the angles.
[0160] Since the protein A adsorbent has a property of selectively
coupling to the Fc region of IgG, this example is suitable for
preparation of a reference sample by removing only IgG from a
sample.
Example 2
[0161] This example shows a chip for optical analysis for a passage
without a bypass passage as shown in FIGS. 2A and 2B, for which a
modified dextran microparticle adsorbent is used as an
adsorbent.
[0162] A cellulose acetate solution obtained by dissolving
cellulose acetate in an organic solvent and modified dextran
microparticles (obtained from the microparticlation of
methacrylate-modified dextran by radical polymerization) (diameter
15 to 30 .mu.m) were mixed to prepare a cellulose acetate-dextran
microparticle mixture solution. A gold thin film (thickness 50 nm)
was formed on a BK7 glass substrate by vapor-deposition. A resist
membrane or a tape was applied onto this gold thin film to form a
pattern in which a portion of the gold thin film corresponding to a
passage was exposed. At this time, an adsorption region of the
passage on the metal thin film was prevented from being exposed,
and the cellulose acetate solution was prevented from being
deposited on the adsorption region. Subsequently, the patterned
gold/glass substrate was mounted on a spin coater, the previously
prepared cellulose acetate solution was added dropwise, Lhen the
substrate was rotated, and the organic solvent was evaporated to
form a white membrane-form passage. Subsequently, the previously
prepared cellulose acetate-dextran microparticle mixture solution
was added dropwise onto the adsorption region and dried, and the
adsorption region was formed so as to connect to the previously
formed white membrane-form passage. Thus, a passage having a length
of 10 mm, a width of 1 mm, and a thickness of 1 .mu.m was
formed.
[0163] Subsequently, a small amount of a phosphate buffer solution
containing 100 .mu.gmL.sup.4 anti-human IgG (0.1 M, pH 6.8) was
added dropwise onto a portion of the passage corresponding to a
measurement region (a portion corresponding to line 3 in FIGS. 2A
and 2B) and dried to immobilize the antibody. Similarly, a
phosphate buffer solution containing 100 .mu.gmL.sup.-1 anti-goat
IgG was added dropwise onto a portion adjacent to the region on
which anti-human IgG was immobilized (a portion corresponding to
line 2 in FIGS. 2A and 2B) and dried to immobilize a reference
antibody.
[0164] Subsequently, a phosphate buffer solution containing 4
mgmL.sup.-1 bovine serum albumin was prepared as a blocking agent
solution, and this solution was added dropwise onto the measurement
region and the reference region on which the antibodies were
immobilized (lines 3 and 2 in FIGS. 2A and 2B) and further another
region in the reference region (a portion corresponding to line 1
in FIGS. 2A and 2B) and dried.
[0165] The prepared chip for optical analysis was mounted on a
prism of a surface plasmon resonance measurement device via a
matching oil. A phosphate buffer solution containing human IgG was
added dropwise onto a sample introduction section of the passage of
the chip for optical analysis as a liquid sample. Human IgG, a
substance to be measured in the liquid sample, is adsorbed to
dextran microparticles in the adsorption region, the phosphate
buffer solution not containing human IgG reaches to the observation
region, and SPR is observed. The angle of SPR observed in the
reference sample not containing human TgG serves as a base level
for measurement.
[0166] Subsequently, after the adsorbent region is saturated by the
liquid sample is further added dropwise, or substance to be
measured is prevented from being adsorbed to the adsorbent by
protecting (or covered) the adsorption region with a plastic or the
like, a large volume of the liquid sample is introduced through the
sample introduction section, and thus the liquid sample containing
human IgG reaches the measurement region, and human IgG binds
selectively to anti-human IgG in the measurement region. On the
other hand, the both regions of the region on which anti-goat TgG
was immobilized and the region on which only a blocking agent was
immobilized in the reference region do not bind to human IgG.
Therefore, at this stage, the angle of SPR increases only in the
measurement region (a portion corresponding to line 3 in FIGS. 2A
and 2B), and the angle of SPR only slightly increases in the
reference region (lines 2 and 1 in FIGS. 2A and 2B) as shown in
FIG. 7. The difference between the measurement region (line 3) and
the region on which anti-goat IgG was immobilized (line 2) in the
reference region represents selective binding of human IgG to
anti-human IgG, and human IgG can be quantified from these changes
over time in the increase in the angles.
[0167] Since dextran microparticles adsorb proteins other than IgG,
this example is suitable for preparation of reference samples in
which various proteins are free. Specifically, dextran
microparticles can be used as adsorbents when proteins other than
IgG, a substance to be measured, are to be removed, or proteins
other than IgG are to be measured as substances to be measured.
Example 3
[0168] This example is a chip for optical analysis with a passage
having a bypass passage as shown in FIGS. 3A and 3B using a protein
A adsorbent as an adsorbent.
[0169] Cellulose acetate was dissolved in an organic solvent to
prepare a cellulose acetate solution. A gold thin film (thickness
50 nm) was formed on a BK7 glass substrate by vapor-deposition. A
resist membrane or a tape was applied onto this gold thin film, and
a pattern in which portions of the gold thin film corresponding to
the main passage and the bypass passage were exposed was formed.
The patterned gold/glass substrate was mounted on a spin coater,
the previously prepared cellulose acetate solution was added
dropwise, then the substrate was rotated, and the organic solvent
was evaporated to form white membrane-form main passage and bypass
passage. Subsequently, a buffer solution of protein A was prepared
and added dropwise onto an adsorption region of the passage, and
protein A was adsorbed to the passage to form the adsorption
region. An aqueous solution of carbodiimide was added dropwise onto
the region to which protein A was adsorbed and dried to activate
the region beforehand. Thus, a passage having a length of 10 mm, a
width of 1 mm, a thickness of 1 .mu.m was formed with a bypass
passage having a total length of 8 mm.
[0170] Subsequently, a small volume of a phosphate buffer solution
containing 100 .mu.gmL.sup.-1 anti-human IgG (0.1 M, pH 6.8) was
added dropwise onto a portion corresponding to a measurement region
of the main passage (a portion corresponding to line 3 in FIGS. 3A
and 3B) and dried to immobilize the antibody. Similarly, a
phosphate buffer solution containing 100 .mu.gmL.sup.-1 anti-goat
IgG was added dropwise to a portion adjacent to the region in which
anti-human IgG was immobilized (a portion corresponding to line 2
in FIGS. 3A and 3B) and dried to immobilize a reference
antibody.
[0171] Subsequently, a phosphate buffer solution of 4 mgmL.sup.-1
bovine serum albumin was prepared as a blocking agent solution, and
this solution was added dropwise onto the measurement region and
the reference region in which the antibodies were immobilized
(lines 3 and 2 in FIGS. 3A and 3B) and further another region in
the reference region (a portion corresponding to line 1 in FIGS. 3A
and 3B).
[0172] The prepared chip for optical analysis was mounted on a
prism of a surface plasmon resonance measurement device via a
matching oil. The phosphate buffer solution containing human IgG
was added dropwise onto the sample introduction section of the
passage of the chip for optical analysis as a liquid sample. Human
IgG that is a substance to be measured in the liquid sample is
adsorbed to protein A in the adsorption region, the phosphate
buffer solution not containing human IgG reaches the observation
region, and SPR is observed. The SPR angle observed in this
reference sample not containing human IgG serves as a base level
for measurement.
[0173] Meanwhile, the liquid sample containing human IgG, which was
allowed to flow through the bypass passage, reaches the measurement
region after the reference sample reaches the measurement region,
and human IgG selectively binds to anti-human IgG in the
measurement region. On the other hand, the both regions of the
region on which anti-goat IgG was immobilized and the region on
which only the blocking agent in the reference region was
immobilized do not bind to human IgG. Therefore, at this stage, the
SPR angle increases only in the measurement region (a portion
corresponding to line 3 in FIGS. 3A and 3B), and the SPR angle only
slightly increases in the reference region (a portion corresponding
to lines 2 and 1 in FIGS. 3A and 3B) as shown in FIG. 7. A
difference between the measurement region (line 3) and the region
on which anti-goat IgG was immobilized in the reference region
(line 2) represents selective binding of human IgG to anti-human
IgG, and human IgG can be quantified from these changes over time
in the increase in the angles.
[0174] Since the sample containing a substance to be measured
reaches the observation region via the bypass passage, when a
sample with low viscosity which progresses in the passage rapidly
is to be measured, a time lag can be adjusted by providing the
bypass passage shown in this example, so that the liquid sample
containing the substance to be measured reaches the observation
region after the reference sample reaches the observation region.
Furthermore, since a protein A adsorbent has a property of
selectively coupling to the Fc region of IgG as shown in this
example, this example is suitable for preparation of a reference
sample by removing only IgG from the sample.
Example 4
[0175] This example is a chip for optical analysis with a passage
having a bypass passage as shown in FIGS. 3A and 3B using a dextran
microparticle adsorbent as an adsorbent.
[0176] A cellulose acetate solution was prepared by dissolving
cellulose acetate in an organic solvent, and this cellulose acetate
solution and modified dextran microparticles (obtained from the
microparticlation of methacrylate-modified dextran by radical
polymerization) (diameter 15 to 30 .mu.m) were mixed to prepare a
cellulose acetate-dextran microparticle mixture solution. A gold
thin film (thickness 50 nm) was formed on a BK7 glass substrate by
vapor-deposition. A resist membrane or a tape was applied onto this
gold thin film, and a pattern in which a portion of the gold thin
film corresponding to a passage was exposed was formed. At this
time, a portion corresponding to an adsorption region of the
passage on the metal thin film was prevented from being exposed,
and the cellulose acetate solution was prevented from being
deposited on the adsorption region. Subsequently, the patterned
gold/glass substrate was mounted on a spin coater, the previously
prepared cellulose acetate solution was added dropwise, then the
substrate was rotated, and the organic solvent was evaporated to
form a white membrane-form passage. Subsequently, the previously
prepared cellulose acetate-dextran microparticle mixture solution
was added dropwise onto the adsorption region and dried to form the
adsorption region so as to connect to the previously formed white
membrane-form passage. Thus, a passage having a length of 10 mm, a
width of 1 mm, and a thickness of 1 .mu.m was formed with a bypass
passage having a full length of 8 mm.
[0177] Subsequently, a small amount of a phosphate buffer solution
containing 100 .mu.gmL.sup.-1 anti-human IgG (0.1 M, pH 6.8) was
added dropwise onto a measurement region of the main passage (a
portion corresponding to line 3 in FIGS. 3A and 3B) and dried to
immobilize the antibody. Similarly, a phosphate buffer solution
containing 100 .mu.gmL.sup.-1 anti-goat IgG was added dropwise onto
a portion adjacent to the region on which anti-human IgG was
immobilized (a portion corresponding to line 2 in FIGS. 3A and 3B)
and dried to immobilize a reference antibody.
[0178] Subsequently, a phosphate buffer solution of 4 mgmL.sup.-1
bovine serum albumin was prepared as a blocking agent solution, and
this solution was added dropwise onto the measurement region and
the reference region on which the antibody was immobilized (lines 3
and 2 in FIGS. 3A and 3B) and further another region in the
reference region (a portion corresponding to line 1 in FIGS. 3A and
3B) and dried.
[0179] The prepared chip for optical analysis was mounted on a
prism of a surface plasmon resonance measurement device via a
matching oil. The phosphate buffer solution containing human IgG
was added dropwise onto the sample introduction section of the
passage of the chip for optical analysis as a liquid sample. Human
IgG, a substance to be measured in the liquid sample, is adsorbed
to protein A in the adsorption region, the phosphate buffer
solution not containing human IgG reaches the observation region,
and SPR is observed. The SPR angle observed in this reference
sample not containing human IgG serves as a base level for
measurement.
[0180] Meanwhile, the liquid sample containing human IgG, which was
allowed to flow through the bypass passage, reaches the measurement
region after the reference sample reaches the measurement region,
and human IgG selectively binds to anti-human IgG in the
measurement region. On the other hand, the both regions of the
region on which anti-goat IgG was immobilized and the region on
which only the blocking agent was immobilized in the reference
region do not bind to human IgG. Therefore, at this stage, the SPR
angle increases only in the measurement region (a portion of line 3
in FIG. 2) and the SPR angle only slightly increases in the
reference region (portions corresponding to lines 2 and 1 in FIGS.
3A and 3B) as shown in FIG. 7. A difference between the measurement
region (line 3) and the region on which anti-goat IgG was
immobilized in the reference region (line 2) represents selective
binding of human IgG to anti-human IgG, and human IgG can be
quantified from these changes over time in the increase in the
angles.
[0181] Since the sample containing a substance to be measured
reaches the observation region via the bypass passage, when a
sample with low viscosity which progresses in the passage rapidly
is to be measured, a time lag can be adjusted by providing the
bypass passage shown in this example, so that the liquid sample
containing the substance to be measured reaches the observation
region after the reference sample reaches the observation region.
Furthermore, since the dextran microparticles adsorb proteins other
than IgG in this example, it is suitable for preparation of a
reference sample in which various proteins are free. Specifically,
when proteins other than IgG, a substance to be measured, are to be
removed, or proteins other than IgG are substances to be measured,
dextran microparticles can be used as an adsorbent.
Example 5
[0182] This example is intended to determine what is suitable as a
combination of substances having molecular selectivity used in the
measurement region and the reference region.
[0183] A cover seal 1034 provided with two openings 1030, 1032 was
attached onto a BK7 glass substrate (length 16 mm.times.width 16
mm) on which a gold thin film (thickness about 50 nm) was formed by
vapor-deposition as shown in FIG. 18 (b). A phosphate buffer
solution of anti-human IgG(Fc) extracted from a goat and a
phosphate buffer solution of anti-rabbit IgG(Fc) extracted from a
goat were added dropwise into these openings 1030 and 1032,
respectively, and allowed to stand at room temperature for 20 min
or longer. After this process, the solution was sucked, and the
cover seal was peeled off to prepare a chip I.
[0184] Similarly, using a gold/glass substrate with a cover seal
provided with two openings 1030, 1032, a phosphate buffer solution
of anti-human IgG extracted from a goat was added dropwise into the
opening 1030, allowed to stand at room temperature as described
above, and subjected to a posttreatment to prepare chip II, a
phosphate buffer solution of anti-human IgG(Fc) extracted from a
goat was added dropwise into the opening 1030, a phosphate buffer
solution of anti-human IgG(Fab) extracted from a goat was added
dropwise into the opening 1032, and these solutions were treated in
the same manner as described above to prepare chip III, and a
phosphate buffer solution of anti-human IgG (Fc) extracted from a
goat was added dropwise in the opening 1030, and a phosphate buffer
solution of anti-enterotoxin IgG(Fc) extracted from a rabbit was
added dropwise in to the opening 32, and these solutions were
treated in the same manner as described above to prepare chip
IV.
[0185] Each of the prepared chips was mounted on a surface plasmon
resonance measurement device (Patent Document 1), and a flow cell
made of PDMS was attached on top of the chip as shown in FIG. 21. A
liquid sample supplied from a micro syringe pump is introduced into
the flow cell via a tube. Furthermore, the device was set, so that
the type of a liquid sample could be switched instantly by using a
liquid switch in combination.
[0186] A portion corresponding to the opening 1030 on the substrate
was used as a measurement region 1003, and the portion
corresponding to the opening 1032 was used as a reference region
1004. When a surface plasmon resonance is measured in these two
regions, resonance angles (.theta.(measurement region),
.theta.(reference region)) can be obtained from the measurement
results as shown in FIG. 22. When a change in these resonance
angles over time is measured, {.theta.(measurement
region)-.theta.(reference region)}/time [degs.sup.-1] represented
by the difference of these resonance angles represents a difference
between the two regions in activity of a substance having molecular
selectivity.
[0187] When commercially available cow's milk is introduced as a
liquid sample containing impurities into the chip set using a flow
cells, lipids, casein, proteins, and the like contained in the
cow's milk are adsorbed and accumulated in an observation section
on which an antibody is immobilized and the gold surface on which
the antibody is not immobilized. When a time to allow cow's milk to
flow is extended, a resonance angle measured in the surface plasmon
resonance device is increased.
[0188] When human IgG antigen, which is not contained in cow's
milk, is mixed in cow's milk as a substance to be measured, the
increasing rate [degs.sup.-1] of .theta.(measurement region) in the
measurement region on which the anti-human IgG is immobilized is
expected to exceed the increasing rate [degs.sup.-1] of
.theta.(reference region). When an antigen having a low
concentration at the level of several nanograms per mL is measured,
a signal in the order of 10.sup.-6[degs.sup.-1] is obtained.
Therefore, the baseline needs to be limited to the level of
10.sup.-6 [degs.sup.-1] or lower. In this example, the baseline is
a difference generated when cow's milk not containing anti-human
IgG, that is, cow's milk alone is introduced to the chip.
[0189] When measurement was made using the chips II, III, and IV,
the baseline was changed in the range of 10.sup.-5 to 10.sup.-4
[degs.sup.-1]. On the other hand, the baseline was limited to
10.sup.-6 [deg-s.sup.-1] or lower using the chip I (see FIG.
23).
[0190] Thus, by using the Fc regions, which have an equal physical
size as IgG antibody, of IgG of the same line which are extracted
from the same animal and are different only in selectivity as
antibodies for both the measurement region and the reference
region, changes in the baseline caused by adsorption and
accumulation of impurities as a noise when a liquid sample
containing many impurities is measured can be minimized.
Example 6
[0191] Using a gold/glass substrate having a cover seal provided
with two openings 1030, 1032, a phosphate buffer solution of
anti-human IgG extracted from a goat was added dropwise into these
two openings 1030, 1032, and only a portion corresponding to the
reference region of the opening 1032 was irradiated with an
electron beam to decrease selectivity of the antibody. Otherwise,
the same procedure as in Example 5 was performed to obtain a chip
for optical analysis V. This chip for optical analysis V was
mounted on a surface plasmon resonance device as in Example 5, a
flow cell was set, and cow's milk was introduced to measure a
surface plasmon resonance. The change in the baseline of
differential measurement between the measurement region and the
reference region was limited to 10.sup.-6 [degs.sup.-1] or
lower.
Example 7
[0192] To prepare a substance having molecular selectivity for the
reference region 1004, a phosphate buffer solution of anti-human
IgG extracted from a goat was heated in a thermostatic bath at
40.degree. C. for 1 h, and the temperature was returned to room
temperature. By this procedure, a phosphate buffer solution of
inactivated anti-human IgG was obtained.
[0193] Subsequently, using a gold/glass substrate having a cover
seal provided with two openings 1030, 1032, a phosphate buffer
solution of anti-human IgG extracted from a goat was added dropwise
into the opening 1030, and a phosphate buffer solution of
previously prepared and inactivated anti-human IgG was added
dropwise onto a portion corresponding to the reference region of
the opening 1032. Otherwise, the same procedure as in Example 5 was
performed to obtain a chip for optical analysis VI. This chip for
optical analysis VI was mounted on a surface plasmon resonance
device in the same manner as in Example 5, a flow cell was set, and
cow's milk was introduced to measure a surface plasmon resonance.
The change in the baseline of differential measurement between the
measurement region and the reference region was limited to
10.sup.-6 [degs.sup.-1] or lower.
Example 8
[0194] Three openings for forming such a metal thin film as shown
in FIG. 19 (a), openings for forming pad sections 1044, 1045, and a
mask having openings for joining sections 1046, 1047 were provided
on a BK7 glass substrate. Gold thin films 1048, 1049, 1050 were
formed through openings by means such as vapor-deposition.
Subsequently, the mask was removed, and a cover seal having
openings 1051, 1052 was applied to the substrate so that the
openings 1051, 1052 should match portions of the gold thin films
1048, 1049 formed at the positions of the openings (FIG. 19 (b)). A
phosphate buffer solution of anti-human IgG (Fc) extracted from a
goat was added dropwise into these openings 1051, 1052, allowed to
stand at room temperature for 20 min, and then removed, and the
cover seal was peeled off.
[0195] Subsequently, as shown in FIG. 19 (c), a cover seal having
an opening 1053 is attached with which a part of the joining
sections 1046, 1047 is covered, the pad sections 1044, 1045 are
exposed, and the gold thin film 1049 on which anti-human IqG(Fc)
extracted from a goat is immobilized and a portion of the gold thin
film 1050 on which anti-human IgG(Fc) extracted from a goat is not
immobilized are exposed. Here, the openings from which the gold
thin film 1049 and the gold thin film 1050 are exposed are in a
well shape and can hold a solution. A phosphate buffer solution was
added dropwise into the well-shaped opening portions of the
substrate formed in this manner. Subsequently, as shown in FIG. 20,
a silver-silver chloride reference electrode was arranged so that a
tip thereof should be brought into contact with a phosphate buffer
solution in the well-shaped openings and connected to a
potentiostat. The tip was connected to the potentiostat using the
portion of the gold thin film 1049 as an action electrode and the
portion of the gold thin film 1050 as a counter electrode.
Subsequently, a potential cycle of sweeping a potential applied to
the action electrode from -0.3 to 1.2 [V vs. silver-silver chloride
reference electrode] and returning from 1.2 to -0.3 [V vs.
silver-silver chloride reference electrode] was given using the
potentiostat and a function generator. By application of this
potential cycle, anti-human IgG (Fc) extracted from a goat on the
metal thin film 1049 was inactivated. Finally, the reference
electrode, the phosphate buffer solution, the cover seal, the
potentiostat, the function generator, and the like were removed to
obtain a chip for optical analysis VII.
[0196] In the same manner as in Example 5, this chip for optical
analysis VII was mounted on a surface plasmon resonance device, a
flow cell was set, cow's milk was introduced, and a surface plasmon
resonance was measured. The change in the baseline in differential
measurement between the measurement region and the reference region
was limited to 10.sup.-6 [degs.sup.-1] or lower.
Example 9
[0197] Using a gold/glass substrate having a cover seal provided
with two openings 1030, 1032, a phosphate buffer solution of
anti-human IgG extracted from a goat was added dropwise into these
two openings 1030, 1032, and a reference buffer at pH 1.68
(Nacarai) was added dropwise into a portion corresponding to the
reference region of the opening 1032 to decrease selectivity of the
antibody. Otherwise, the same procedure as in Example 5 was
performed to obtain a chip for optical analysis VIII. This chip for
optical analysis VIII was mounted on a surface plasmon resonance
device in the same manner as in Example 5, a flow cell was set, and
cow's milk was introduced, and a surface plasmon resonance was
measured. The change in the baseline of differential measurement
between the measurement region and the reference region was limited
to 10.sup.-6 [degs.sup.-1] or lower.
Example 10
[0198] Phosphate buffer solutions each containing DNA having the
following strand A (SEQ ID NO: 1) or strand B (SEQ ID NO: 2) were
prepared. Strand A is DNA for the measurement region, and Strand B
is DNA for the reference region.
TABLE-US-00001 Strand A: (SEQ ID NO: 1) 5'-CCT CTG ACT TCA ACA GCG
ACA CT-3' Strand B: (SEQ ID NO: 2) 5'-CCT CAG ACT TCA ACA GGG ACA
CT-3'
[0199] Using a gold/glass substrate having a cover seal provided
with two openings 1030, 1032, a phosphate buffer solution
containing strand A was added dropwise into the opening 1030, a
phosphate buffer solution containing strand B was added dropwise
into the opening 1032, the solutions were sucked in the same manner
as in Example 5, and the DNA molecules were immobilized on the gold
thin film to obtain a chip for optical analysis X. This chip for
optical analysis X was mounted on a surface plasmon resonance
device in the same manner as in Example 5, a flow cell was set, and
cow's milk was introduced, and a surface plasmon resonance was
measured. The change in the baseline of differential measurement
between the measurement region and the reference region was limited
to 10.sup.-6 [degs.sup.-1] or lower.
Example 11
[0200] A chip for optical analysis having a plurality of
observation sections was prepared as shown in FIG. 17. A phosphate
buffer solution of anti-human IgG(Fc) extracted from a goat and a
phosphate buffer solution of anti-rabbit IgG(Fc) extracted from a
goat were added dropwise onto the measurement region and the
reference region of the observation section 1010, respectively.
Furthermore, DNA strands A and B used in Example 10 were added
dropwise onto the measurement region and the reference region,
respectively, in the observation section 1011. The solution in each
region was sucked, and both the antibodies and the DNA molecules
were immobilized to obtain a chip for optical analysis XI. This
chip for optical analysis XI was mounted on a surface plasmon
resonance device in the same manner as in Example 5, a flow cell
was set, and cow's milk was introduced, and a surface plasmon
resonance was measured. The changes in the baselines in
differential measurement between the measurement region and the
reference region on which the antibodies were immobilized and the
measurement region and the reference region on which the DNA
molecules were immobilized were both limited to 10.sup.-6
[degs.sup.-1] or lower.
Example 12
[0201] Using the chip for optical analysis I prepared in Example 5,
a liquid sample comprising cow's milk containing 50 ngmL.sup.-1 of
human IgG antigen was introduced into such a measurement device
provided with a surface plasmon resonance device and a flow cell as
explained in Example 5 to measure a surface plasmon resonance. As a
result, as compared with the level of the baseline, a response with
a clearly large slope was obtained for a signal represented by
{.theta.(measurement region)-.theta.(reference region)} (see FIG.
24). The level of the baseline was obtained by measuring cow's milk
not containing human IgG before the introduction of the liquid
sample.
[0202] The above-mentioned results suggest that more human IgyG
antigen molecules were bound to the measurement region on which
anti-human IgG that had not been inactivated was immobilized. On
the other hand, the above-mentioned results suggest that
selectivity of the inactivated anti-human IgG immobilized on the
reference region to human IgG antigen was decreased as compared
with the measurement region.
[0203] Subsequently, using the chips for optical analysis V, VI,
VII, and VIII prepared in Examples 6 to 9, a liquid sample
comprising cow's milk containing 50 ngmL.sup.-1 human IgG antigen
was similarly measured for a surface plasmon resonance. As a result
of measurement, a response having the same slope as shown in FIG.
24 was obtained. This result suggests that more human IgG antigen
molecules were bound to the measurement region on which anti-human
IgG antibody that had not been inactivated or subjected to protein
engineering was immobilized. On the other hand, the above-mentioned
result suggests that selectivity of the inactivated anti-human IgG
immobilized on the reference region to human IgG antigen was
decreased as compared with the measurement region.
[0204] Subsequently, cow's milk containing 1 nmolmL.sup.-1 DNA
comprising strand C (SEQ ID NO: 3), which forms a specific bond
with the strand A, was prepared, and a surface plasmon resonance
was measured using the chip for optical analysis X as described
above.
TABLE-US-00002 Strand C: (SEQ ID NO: 3) 3'-GGA GAC TGA AGT TGT CGC
TGT GGG TG-5'
[0205] As a result, a response with a slope greater than the level
of the baseline obtained from cow's milk not containing DNA,
3.times.10.sup.-5 [degs.sup.-1], was obtained.
[0206] Furthermore, using the chip for optical analysis XI on which
antibodies and DNA molecules were immobilized, a surface plasmon
resonance of a cow's milk sample containing 50 ngmL.sup.-1 human
IgG antigen and DNA comprising the strand C was similarly measured.
As a result, a response with a slope which was greater than the
baseline level and the same as shown in FIG. 24 was obtained for a
signal represented by {.theta.(measurement
region)-.theta.(reference region)} in the observation section on
which the antibodies were immobilized, and 3.times.10.sup.-5
[degs.sup.-1] that is a response similar to that described above
was obtained in the observation section on which DNA molecules were
immobilized.
INDUSTRIAL APPLICABILITY
[0207] The chip for optical analysis of the present invention can
be utilized in an optical measurement method using a total
reflection optical system that can directly observe binding of a
molecule and an immobilized substance having molecular selectivity.
Sequence CWU 1
1
3123DNAArtificial SequenceThis sequence was artificially
synthesized. 1cctctgactt caacagcgac act 23223DNAArtificial
SequenceThis sequence was artificially synthesized. 2cctcagactt
caacagggac act 23326DNAArtificial SequenceThis sequence was
artificially synthesized. 3ggagactgaa gttgtcgctg tgggtg 26
* * * * *