U.S. patent application number 10/798001 was filed with the patent office on 2005-03-17 for substrate for bio-microarray and bio-microarray.
Invention is credited to Hattori, Hideshi.
Application Number | 20050059169 10/798001 |
Document ID | / |
Family ID | 33287335 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050059169 |
Kind Code |
A1 |
Hattori, Hideshi |
March 17, 2005 |
Substrate for bio-microarray and bio-microarray
Abstract
For the purpose of improving the accuracy of a variety analyses
using a bio-microarray, it is a main object of the invention to
provide a bio-microarray capable of producing relatively high
signal intensity from a fluorescent molecule and capable of having
improved quantification performance and to provide a substrate for
bio-microarray. In order to achieve the object, the invention
provides a substrate for bio-microarray characterized by having a
reflection-suppressing function.
Inventors: |
Hattori, Hideshi; (Tokyo-to,
JP) |
Correspondence
Address: |
LADAS & PARRY LLP
224 SOUTH MICHIGAN AVENUE
SUITE 1200
CHICAGO
IL
60604
US
|
Family ID: |
33287335 |
Appl. No.: |
10/798001 |
Filed: |
March 11, 2004 |
Current U.S.
Class: |
436/518 |
Current CPC
Class: |
B01J 19/0046 20130101;
B01J 2219/00637 20130101; B01J 2219/00659 20130101; B01J 2219/00641
20130101; B01J 2219/0054 20130101; B01J 2219/00605 20130101; C40B
70/00 20130101; B01J 2219/00527 20130101; B01J 2219/00596 20130101;
B01J 2219/00612 20130101 |
Class at
Publication: |
436/518 |
International
Class: |
G01N 033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 14, 2003 |
JP |
2003-070639 |
Claims
What is claimed is:
1. A substrate for bio-microarray having a reflection-suppressing
function.
2. The substrate for bio-microarray according to claim 1, wherein
the substrate for bio-microarray comprises a substrate and at least
one of an anti-reflection layer and a light-absorbing layer formed
on a surface of the substrate, whereby the substrate for
bio-microarray has the reflection-suppressing function.
3. The substrate for bio-microarray according to claim 2, wherein
at least one of the anti-reflection layer and the light-absorbing
layer is formed in a pattern.
4. The substrate for bio-microarray according to claim 1, wherein
the substrate for bio-microarray comprises a substrate having a
fine uneven structure or a fine porous structure at a surface of
the substrate.
5. The substrate for bio-microarray according to claim 2, wherein
the anti-reflection layer has a fine uneven structure or a fine
porous structure.
6. The substrate for bio-microarray according to claim 2, wherein
the light-absorbing layer has a fine uneven structure or a fine
porous structure.
7. The substrate for bio-microarray according to claim 1, wherein
the substrate for bio-microarray comprises a substrate and an
immobilization layer for immobilizing a probe biomolecule, which is
formed in a pattern on the substrate.
8. The substrate for bio-microarray according to claim 1, wherein
the substrate for bio-microarray comprises a substrate and a mark
for positional detection, which is formed on the substrate.
9. A bio-microarray, comprising the substrate for bio-microarray
according to claim 1 and a probe biomolecule immobilized on the
substrate.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to a bio-microarray having probe
biomolecules such as DNA, peptide, protein, polysaccharide, cell,
and tissue, which are arranged in an array, and also relates to a
substrate for bio-microarray.
[0003] 2. Description of the Related Art
[0004] In recent years, the target of development of
bio-microarrays (also referred to as biochips) has been expanded
and thus includes not only DNA microarrays, which have been used in
genome analysis, but also microarrays that load any other
biomolecules such as peptide, protein, polysaccharide, cell, and
tissue.
[0005] The bio-microarray analysis method generally includes the
processes of introducing a fluorescent molecule as a label into a
sample and analyzing light emission from the fluorescent molecule,
because such a process is appreciated as having a high sensitivity
and a variety of available labels. Examples of the analytical
apparatus according to this method include: a high accuracy type
apparatus that uses the principle of confocal microscope and
performs a spot-by-spot analysis on the bio-microarray; and a high
throughput type apparatus that uses a charge-coupled device array
or the like as a detector and analyzes a plurality of spots in a
specific region at a time. In this method, for example, the data
signal-to-noise ratio can be reduced by such a factor as a noise
generated at the time of data amplification/conversion, the
reflection of excitation light (incident light), dusts on the
substrate, an unevenness in the surface treatment state of a glass
substrate, and a background fluorescence. In particular, the
reflection of excitation light is a remarkable cause of the
reduction in accuracy that can come from the substrate.
[0006] On the other hand, an increase in the detected signal
intensity requires sufficient absorption of the excitation light
into the label of the fluorescent molecule, and therefore, in some
cases, suppression of the reflection of the excitation light can
reduce the analysis accuracy depending on the intensity of the
excitation light.
[0007] Thus, it is desirable that the user should use different
substrates so that the reflection of excitation light can be
reduced or increased depending on the intensity of the excitation
light. Specifically, however, substrates produced with no optical
processing and substrates having the function of increasing the
reflection are only commercially available now. In some cases, such
substrates cannot provide sufficient analytical accuracy, depending
on the intensity of the excitation light.
SUMMARY OF THE INVENTION
[0008] For the purpose of improving the accuracy of a variety of
bio-microarray analyses, it is a main object of the invention to
provide a bio-microarray capable of producing relatively high
signal intensity from a fluorescent molecule and capable of having
improved quantification performance and to provide a substrate for
bio-microarray.
[0009] The invention provides a substrate for bio-microarray
characterized by having a reflection-suppressing function. The
inventive substrate for bio-microarray has the function of
suppressing reflection and thus can increase the analysis accuracy
through a relative increase in the signal intensity of the
fluorescent molecule by the suppression of the reflection of the
excitation light.
[0010] In the invention, at least one of an anti-reflection layer
and a light-absorbing layer is preferably formed on the surface of
the substrate so as to produce the reflection-suppressing function.
The anti-reflection layer can suppress the reflection by increasing
the transmittance of the excitation light, or the light-absorbing
layer can suppress the reflection by absorbing the excitation
light. The reflection-suppressing function can easily be given to
the microarray substrate by the formation of the anti-reflection
layer or the light-absorbing layer.
[0011] In the invention, at least one of the anti-reflection layer
and the light-absorbing layer is preferably formed in a pattern.
When the anti-reflection layer or the light-absorbing layer having
the reflection-suppressing function is formed in a pattern, the
region having the reflection-suppressing function can be selected.
In such a structure, the signal intensity of the fluorescent
molecule can be increased while the reflection of the excitation
light can be suppressed, so that the analysis accuracy can further
be improved.
[0012] In the invention, the surface of the substrate also
preferably has a fine uneven structure or a fine porous structure.
Such a structure can have a high density of probe biomolecules
immobilized on the surface of the substrate and thus can increase
the detection accuracy.
[0013] In addition, the anti-reflection layer preferably has a fine
uneven structure or a fine porous structure, or the light-absorbing
layer preferably has a fine uneven structure or a fine porous
structure. When the anti-reflection layer or the light-absorbing
layer has a fine uneven structure or a fine porous structure, such
a layer formed on the substrate can provide both the effect of
suppressing the reflection and the effect of increasing the density
of probe biomolecules immobilized on the surface of the substrate,
so that the detection accuracy can easily be improved.
[0014] In the invention, an immobilization layer for immobilizing
the probe biomolecule on the substrate is preferably formed in a
pattern. Such a structure can prevent the diffusion of the probe
biomolecule and thus can provide a higher packing density. Such a
structure can also prevent the adsorption of unnecessary probe
biomolecules and thus can further increase the analysis
accuracy.
[0015] In the invention, a mark for positional detection is
preferably formed on the substrate. Such a structure can provide an
improvement in workability and a reduction in error rate in the
process of immobilizing the probe biomolecule on the substrate and
in the analysis process thereafter.
[0016] The invention also provides a bio-microarray characterized
by comprising any of the above-mentioned substrates for
bio-microarray and a probe biomolecule immobilized on the
substrate. The bio-microarray using the inventive substrate for
bio-microarray can relatively increase the signal intensity of the
fluorescent molecule and can have good quantification performance,
so that it can produce a high analytical accuracy.
[0017] The inventive substrate for bio-microarray has the
reflection-suppressing function and thus can form a bio-microarray
with a relatively increased signal intensity of a fluorescent
molecule and a high analytical accuracy. An anti-reflection layer,
a light-absorbing layer or a substrate surface each having a
specific structure may be used to provide the
reflection-suppressing function. In such a case, not only the
reflection-suppressing function but also a high packing density of
probe biomolecules can be provided so that a bio-microarray with
good quantification performance can be provided. In a case where
the reflection-suppressing function and the reflection function are
provided in a pattern, the signal intensity of the fluorescent
molecule can be increased while the reflection of unnecessary
excitation light can be suppressed, so that the analysis accuracy
of the bio-microarray can further be improved.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 are schematic diagrams each showing an example of the
inventive substrate for bio-microarray;
[0019] FIG. 2 are schematic diagrams each showing an example of the
anti-reflection layer according to the invention; and
[0020] FIG. 3 are schematic diagrams each showing another example
of the anti-reflection layer according to the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0021] First, a detailed description is provided of the inventive
substrate for bio-microarray and a method of producing the
same.
[0022] A. Substrate for Bio-Microarray
[0023] The inventive substrate for bio-microarray is characterized
by having the reflection-suppressing function and thus has an
advantage that it can relatively increase the signal intensity of a
fluorescent molecule used as a label by suppressing the reflection
of excitation light that excites the fluorescent molecule. First,
such a reflection-suppressing function is described below.
[0024] Reflection-Suppressing Function
[0025] In the invention, the reflection-suppressing function can be
produced by a method of increasing the transmittance of excitation
light, a method of absorbing the excitation light, or the like.
[0026] The method of suppressing the reflection by increasing the
transmittance of excitation light may generally be a method of
forming an anti-reflection layer on a surface but may alternatively
be a method of producing an anti-reflection function by processing
the surface of the substrate. The method of suppressing the
reflection by absorbing excitation light may be a method of forming
a light-absorbing layer on a surface or a method of using a
substrate originally having a light-absorbing function.
[0027] The anti-reflection layer may be formed on the substrate
surface on the side where the probe biomolecule will be immobilized
or on the side opposite to it. The anti-reflection layer is
preferably formed on the surfaces on both sides in terms of
preventing reflection on both sides. Similarly, the anti-reflection
function may be given to the substrate surface on the side where
the probe biomolecule will be immobilized or on the side opposite
to it, and is preferably given to the surfaces on both sides.
[0028] The light-absorbing layer may also be formed on the
substrate surface on the side where the probe biomolecule will be
immobilized or on the side opposite to it. In view of its
characteristics, it may generally be formed on either side surface.
In the case where the substrate itself has the light-absorbing
function, the substrate may entirely has the light-absorbing
function, as in a case where the substrate is entirely black, or
the substrate surface may have the light-absorbing function only on
the side where the probe biomolecule will be immobilized or on the
side opposite to it.
[0029] In the invention, any of these conditions may appropriately
be selected or combined so that the most preferred mode can be used
according to the conditions such as the type of the substrate and
the type of the immobilization layer.
[0030] FIG. 1 each show an example of the substrate for
bio-microarray with the reflection-suppressing function provided by
the above method.
[0031] FIG. 1A shows an example in which an anti-reflection film 2
is formed on a substrate 1, and an immobilization layer 3 for
immobilizing probe biomolecules is formed thereon. FIG. 1B shows an
example in which a light-absorbing layer 4 is formed on the
substrate 1, and the immobilization layer 3 is formed thereon. In
both examples, the function is given to the substrate surface on
the side where the probe biomolecules will be immobilized.
[0032] FIG. 1C shows an example in which the anti-reflection layer
2 is formed on both side surfaces of the substrate 1, and the
immobilization layer 3 is formed on one of the anti-reflection
layers 2. FIG. 1D shows an example in which the anti-reflection
layer 2 is formed on the surface of the substrate 1 on the side
where the probe biomolecule will be immobilized; the immobilization
layer 3 is stacked on the surface thereof; and the light-absorbing
layer 4 is formed on the opposite side surface of the substrate
1.
[0033] When the anti-reflection layer 2 according to the invention
is formed on the surface of the substrate 1 on the side where the
probe biomolecule will be immobilized, the reflection from the
other surface should be suppressed, and thus it is preferred that
the anti-reflection layer 2 or the light-absorbing layer 4 is
formed on the other surface.
[0034] In addition, any other functional layer may also be formed
between the respective layers. For example, an adhesive layer for
improving the adhesion properties may also be formed. The
anti-reflection layer 2 and the light-absorbing layer 4 may each
comprise a single layer or plural layers. Specifically, the
anti-reflection layer 2 comprising plural layers is generally
formed by stacking a low refractive index layer and a high
refractive index layer alternately.
[0035] A description is then provided of the region having such a
reflection-suppressing function (hereinafter, also referred to as a
reflection-suppressing function region).
[0036] In the invention, such a reflection-suppressing function
region may be formed over the entire surface of the substrate, or
may be formed in a pattern to produce higher detection accuracy. In
such a structure, the reflection-suppressing function region may
have any mode of the above combinations, and for example, the
patterned region may be a patterned anti-reflection layer, a
patterned light-absorbing layer, a patterned anti-reflection layer
formed on both side surfaces of the substrate as in the above
example, or the like.
[0037] In the invention, the pattern of the reflection-suppressing
function region may be formed only in a region other than the
region where the probe biomolecule will be immobilized (hereinafter
also referred to as a probe molecule immobilization region) or may
be formed only in such a probe biomolecule immobilization region.
If the excitation light will be applied over the entire surface of
the substrate at the time of detecting fluorescence from the label,
it is preferred that the pattern should be formed only in a region
other than the probe biomolecule immobilization region. It is
because in such a case, the reflection of the excitation light from
the region other than the probe biomolecule immobilization region
can be a cause of a reduction in detection accuracy. On the other
hand, if the excitation light will be applied only to the probe
biomolecule immobilization region, it is preferred that the
reflection-suppressing function region should be patterned only in
the probe biomolecule immobilization region. It is because the
problem of the excitation light reflection could occur in the probe
biomolecule immobilization region.
[0038] In the mode where the reflection-suppressing function region
is formed only in the region other than the probe biomolecule
immobilization region according to the invention, the probe
biomolecule immobilization region may be a reflection function
region having the function of reflecting the excitation light. Such
a structure has an advantage that the accuracy of the detection by
the reflected light from the excitation light can be reduced at the
time of applying the excitation light while the excitation light is
reflected from the probe biomolecule immobilization region so that
the label of the fluorescent molecule can easily be excited. When
the label of the fluorescent molecule emits light, the fluorescent
light is also reflected from the substrate side so that the
fluorescent light can easily be detected and that the detection
accuracy can be increased.
[0039] In such a structure, any reflection function region may be
provided, as long as it has the function of reflecting the
excitation light and the fluorescent light from the label. For
example, such a region is formed by a method of forming a
reflecting layer on the substrate or the like.
[0040] Thus, the probe biomolecule immobilization region may be the
reflection function region while the region other than the probe
biomolecule immobilization region may be the reflection-suppressing
function region. For example, a method of forming such regions
includes the processes of: patterning the reflecting layer on the
substrate surface on the side where the bio-macromolecule will be
immobilized in order to form the reflection function region; and
patterning the light-absorbing layer on the opposite side surface
in order to form the reflection-suppressing function region,
wherein the same photomask is used in the processes; one side
surface is patterned by a positive type photoresist and another
side surface is patterned by a negative type photoresist in the
processes.
[0041] Fine Uneven Structure or Fine Porous Structure
[0042] According to the invention, a fine uneven structure or a
fine porous structure is preferably formed at the substrate surface
on the side where the probe biomolecule will be immobilized, in
order to increase the detection accuracy of the fluorescent light
for use as a label.
[0043] Such a fine uneven or fine porous structure can
significantly increase the surface area for immobilizing the probe
biomolecules. Even if the surface region has the same appearance,
the region formed with the fine uneven or fine porous structure can
immobilize more probe biomolecules so that the detection accuracy
can significantly be increased.
[0044] Such a fine uneven or fine porous structure may be formed in
at least the probe biomolecule immobilization region, and thus may
be formed in a pattern only in the probe biomolecule immobilization
region or may be formed over the entire surface.
[0045] In a particularly preferred mode of the invention, the
anti-reflection layer or the light-absorbing layer has the fine
uneven structure or the fine porous structure. It is because such a
structure can dramatically improve the detection accuracy by both
of the detection accuracy-increasing effect of the anti-reflection
or light-absorbing layer formation and the detection
accuracy-increasing effect of the fine uneven or fine porous
structure formation.
[0046] The fine uneven or fine porous structure of the
anti-reflection or light-absorbing layer may be formed one or both
sides of the substrate. If the structure is formed on one side, it
should preferably formed on the side where the probe biomolecule
will be immobilized, in terms of higher packing density of the
probe biomolecules.
[0047] Anti-Reflection Layer
[0048] Any anti-reflection layer can be used in the invention as
long as it can produce high transmittance of excitation light. The
anti-reflection layer preferably has a refractive index smaller
than that of the substrate and a thickness sufficient for the
prevention of the reflection.
[0049] In the invention, the anti-reflection layer may have a flat
structure but preferably has the fine uneven or fine porous
structure as described above.
[0050] For example, the anti-reflection layer having the flat
structure may be a film formed of a material having a generally low
refractive index, a laminate of a low refractive index layer and a
high refractive index layer as shown above, or the like.
[0051] Examples of the material for the anti-reflection layer with
the flat structure include silicon oxides (for a low refractive
index layer) and niobium oxides, Ti oxides, zirconium oxides, and
ITO (for a high refractive index layer).
[0052] Such a flat anti-reflection layer may be formed by any
conventional method such as a dry method including a vacuum
deposition method, a sputtering method, a CVD method, or the like;
and a coating method.
[0053] Examples of the anti-reflection layer having the fine uneven
or fine porous structure are described with reference to FIGS. 2,
and 3.
[0054] FIGS. 2A and 2B are schematic diagrams each showing an
example of the anti-reflection layer 2 having the fine uneven
structure formed on the substrate 1. As shown in the drawings, the
fine uneven structure may be formed by a method including the
process of processing a flat film by etching or the like so as to
form unevenness or may be formed by arranging fine particles with
any of various shapes in a layer.
[0055] The unevenness may also be formed by a process including the
processes of: applying, to a substrate, a coating liquid comprising
different polymers incompatible with one another and a solvent in
which the polymers are dissolved; drying it to form a
phase-separated film; and then treating the film with a solvent
which can dissolve one polymer but cannot dissolve the other
polymer(s) (a reference material: Walheim et al., Science, Vol.
283, page 520, 1999). The anti-reflection layer having the uneven
structure may also be formed by any general technique such as a
sputtering method, a physical vapor deposition (PVD) method, a
chemical vapor deposition (CVD) method, and a sol-gel coating
method. Particularly when the substrate is made of a plastic, the
uneven anti-reflection structure may be formed by embossing
(imprint process). Specifically, when the substrate is made of
alumina, aluminum with alumina layer at the surface or silicon, the
anti-reflection layer may be formed by applying any known method of
forming porous alumina or porous silicon.
[0056] FIGS. 3A and 3B are schematic diagrams each showing an
example of the anti-reflection layer 2 having the fine porous
structure formed on the substrate 1. As shown in the drawings, the
fine porous structure may be a porous layer or may be a laminate of
plural layers of fine particles with any of various shapes.
[0057] First, a description is provided of the fine uneven
structure. The fine uneven structure for use in the invention may
be formed by a method including the process of processing a flat
layer by etching or the like so as to form unevenness or may be
monolayer arranged by fine particles.
[0058] When fine particles are used to form the fine uneven
structure, their average particle diameter is preferably from 50 nm
to 300 nm, more preferably from 70 nm to 250 nm. The above range is
preferred for the purpose of attaining the function of the
anti-reflection layer. It will be understood that the shape of the
fine particles with such a particle diameter is not limited to a
sphere.
[0059] The average particle diameter is defined by a mean value of
the respective particle diameters measured from electron microscope
images. When the material for use is colloidal, the average
particle diameter is determined by a colloid-scientific particle
diameter measurement method such as a light-scattering method.
[0060] In such a structure, any fine particles may be used as long
as they are transparent in the visible light range and can have a
bulk refractive index smaller than that of the substrate when the
fine particle layer is formed.
[0061] Examples of the applicable inorganic material for the fine
particles include MgF.sub.2 (refractive index 1.38), SiO.sub.2
(refractive index 1.46), AlF.sub.3 (refractive index 1.33-1.39),
CaF.sub.2 (refractive index 1.44), LiF (refractive index
1.36-1.37), NaF (refractive index 1.32-1.34), and ThF.sub.4
(refractive index 1.45-1.5).
[0062] Examples of fine organic material particles include fine
particles of polymers. Specific examples thereof include
crosslinked acrylic fine particles (such as MX series and MR series
both manufactured by Soken Chemical & Engineering Co., Ltd.),
uncrosslinked acrylic fine particles (such as MP series
manufactured by Soken Chemical & Engineering Co., Ltd.),
crosslinked polystyrene fine particles (such as SGP series
manufactured by Soken Chemical & Engineering Co., Ltd.),
uncrosslinked polystyrene fine particles, monodisperse poly(methyl
methacrylate) fine particles with a high degree of crosslinking
(such as MS series and M series both manufactured by Soken Chemical
& Engineering Co., Ltd.), fine particles of any combination
thereof, and fine particles having an introduced functional
group.
[0063] In particular, silica (SiO.sub.2) fine particles or polymer
fine particles are preferably used in the invention.
[0064] On the other hand, the anti-reflection layer having fine
unevenness may be formed by a process including the processes of
forming a flat layer and forming unevenness in the layer by etching
or the like. In such a case, the depth of the uneven structure is
preferably in the range from 80 nm to 250 nm, more preferably from
90 nm to 200 nm, still more preferably from 100 nm to 180 nm. The
above range is preferred for the purpose of prevention from
scattering of incident light. In addition, the pitch of the
unevenness (an average distance between adjacent bumps or dips) is
preferably in the range from 100 nm to 1000 nm, more preferably
from 150 nm to 500 nm. The anti-reflection layer is preferably
provided in the above range.
[0065] A polymer material that is transparent and has no
fluorescent characteristic in the visible light range is preferably
used to form the anti-reflection layer with fine unevenness.
Examples of such a material include (meta)acrylic-based resins,
styrene-based resins, cycloolefin-based resins, polyesters, and
polycarbonates.
[0066] The method of forming the fine uneven structure can broadly
be divided into a subtractive method and an additive method. The
subtractive method includes etching or selectively removing a
material. Examples of the etching method include an oxygen plasma
treatment method and a sandblasting method. Examples of the
selectively removing method include methods using the
phase-separated structure of a polymer and selective solubility of
the polymer as described above. The additive method includes
forming a material into a film with the uneven structure on a
substrate. A dry process includes a sputtering method, a PVD method
and a CVD method. A wet process includes a method of producing a
film with a particle-containing coating liquid by an adsorption
method, a spray method, a dip coating method, or the like.
[0067] A porous layer with the fine porous structure may be
produced with a material having continuous holes whose diameter is
in the range from 3 nm to 90 nm, particularly from 5 nm to 70
nm.
[0068] Such a material may preferably be produced by a method of
forming a laminate of fine particles, such as a layer-by-layer
adsorption method and a particle settling method. The particles are
preferably silica particles or transparent particles such as
polymer particles. The average particle diameter is preferably in
the range from 20 nm to 100 nm, particularly preferably from 30 nm
to 80 nm. Alternatively, the laminate of the fine particles may be
used as a mold for forming the porous structure. For example, such
a method includes the processes of penetrating a low-molecular
material into the pores of the laminate of the particles, allowing
it to react in situ to form a self-supporting material, and then
selectively removing the particles.
[0069] Light-Absorbing Layer
[0070] Any light-absorbing layer may be used as long as it has the
function of absorbing excitation light. In a preferred mode, the
absorption rate of excitation light that reaches the
light-absorbing layer is 50% or more, particularly 90% or more.
[0071] Similarly to the anti-reflection layer, the light-absorbing
layer may have a flat structure or an uneven structure. When the
light-absorbing layer is placed on the immobilization layer side,
it should preferably have the fine uneven structure or the fine
porous structure in terms of higher packing density of probe
biomolecules, similarly to the anti-reflection layer.
[0072] The light-absorbing layer having the flat structure may be
formed by a method of applying coating material, which absorbs
incident light, to a substrate. A colored glass or a colored
plastic may be used as the substrate. The light-absorbing agent
should be a material whose fluorescent spectrum does not overlap
with the fluorescent spectrum of the fluorescent molecule and which
has excitation light absorbing properties. It is particularly
preferred that the light-absorbing agent produces no fluorescence
by the excitation light. Examples of such a material include
graphite and fullerene. The binder may be made of any general
binder resin. The light-absorbing layer having the fine uneven
structure may be formed by an etching, such as sandblasting method,
a colored substrate or substrate with a light-absorbing layer The
light-absorbing layer having the fine porous structure may be
prepared by a method of selectively removing the binder resin, such
as an oxygen plasma method, or by a layer-by-layer adsorption
method or the like.
[0073] Reflecting Layer
[0074] In the invention, it is advantageous that the reflection
function region is formed in a pattern as described above. Such a
reflecting layer may be formed by a method of vapor-depositing, on
a substrate, a metal material such as aluminum, gold and silver or
an organic metal material such as titanium alkoxide, through a
metal mask having the desired opening. Another method includes the
processes of attaching a catalyst in a pattern to a substrate and
performing electroless plating of nickel, copper, gold, or the like
to form the reflecting layer. After the reflecting layer is formed
on the substrate surface by vapor deposition or electroless
plating, it may also be subjected to photoresist lithography and
etching so as to form a desirably patterned reflecting layer (a
reference material: Japanese Patent Application Laid-Open (JP-A)
No. 2002-153272).
[0075] Substrate
[0076] In the invention, the material for the substrate may be
selected so as to satisfy the required characteristics depending on
the use. Examples thereof include a metal such as aluminum, copper,
stainless steel, and zinc; a semiconductor such as silicon; an
inorganic material such as glass; and a film mainly composed of a
polymer material such as polyethylene terephthalate and
polypropylene. A transparent material is generally used, such as
glass and a polymer material. In a preferred mode, the form of the
substrate is flat like a slide glass or has a number of independent
wells like a microwell plate.
[0077] In the invention, for the purpose of providing the substrate
for bio-microarray with the reflection-suppressing function, a
substrate having the reflection-suppressing function by itself may
be used in place of providing an additional layer as described
above. Examples of such a substrate include a substrate whose
surface has the fine uneven structure and a substrate having the
fine porous structure. Such a fine uneven or fine porous structure
may be formed by the method as described above.
[0078] Such a structure may be formed on one or both sides of the
substrate. If the structure will be formed on one side, it should
be preferably formed on the side where the probe biomolecules will
be immobilized, in terms of high-density integration of probe
biomolecules.
[0079] Immobilization Layer
[0080] The immobilization layer for use in the invention is a thin
film that is provided on the substrate for the purpose of
immobilizing the probe biomolecule. For example, such an
immobilization layer is formed by a process including the processes
of: washing the substrate surface with an alkali and rinsing it so
that the surface becomes negatively charged; allowing an
immobilization agent such as poly-L-lysine to adsorb to the surface
so that the surface becomes positively charged; removing
unnecessary poly-L-lysine by a centrifuge; and drying it. There is
another method including the process of chemically vapor-depositing
an active molecule having an amino group on the substrate surface.
For example, the active molecule is aminopropyltriethoxysilane or
the like. There is yet another method of forming an absorption
layer of a polymer material having an aldehyde group or an active
ester group or the like.
[0081] The thickness of the immobilization layer is preferably
within the limits of not ruining the purposes of expanding the
surface area and integrating the probe biomolecules at high density
in the process of forming the anti-reflection or light-absorbing
layer with the uneven structure on the substrate or in the process
of forming the uneven structure in the substrate surface itself as
described above. Specifically, the thickness of the immobilization
layer is preferably 1/3 or less of the average depth of the
unevenness or 1/3 or less of the average pore size, and is more
preferably of the order of monomolecular layer (molecular
adsorption layer) thickness.
[0082] In the invention, it is particularly preferred that regions
having at least two different surface free energies are arranged in
a pattern on the substrate. Specifically, it is preferred that the
immobilization layer should be patterned and that the
immobilization layer and the substrate surface should be different
in surface free energy. Herein, the surface free energy refers to
an energy (erg.multidot.cm.sup.-2) necessary for the generation of
the surface per unit area and may be used as an index to the force
which is exerted on the surface molecules so as to pull them toward
the inside.
[0083] It is also preferred that such regions having different
surface free energies should comprise: a plurality of independent
regions each having a high wettability with respect to the probe
biomolecule and having a first surface free energy; and a
continuous region that is placed around the above regions and has a
low wettability with respect to the probe biomolecule and a second
surface free energy. The difference in surface free energy between
the immobilization layer and the substrate surface is so used that
the loaded probe biomolecules can be prevented from diffusing and
integrated at higher density and that adsorption of unnecessary
probe biomolecules can be prevented so that the analysis accuracy
can further be increased.
[0084] The regions having different surface free energies may have
any of various patterns such as a pattern of a hydrophilic region
and a hydrophobic region, a pattern of an electrically charged
region and an uncharged region, and a pattern of a region capable
of covalently bonding to the probe biomolecule and a region
incapable of covalently bonding to it.
[0085] For example, the patterned regions having different surface
free energies may be formed on the substrate by a method including
the processes of: forming a thin film of an immobilization agent
such as poly-L-lysine as described above; and then forming the
desired pattern using a photocatalyst technique, an etching method,
a photolithography method, or the like. Herein, the photocatalyst
technique refers to the method as disclosed in JP-A Nos.
2002-274077 and 2000-249821.
[0086] Alternatively, a printing method such as a microcontact
printing method, a flexo printing method and a gravure method may
be used to form the patterned regions with different surface free
energies on the substrate.
[0087] Mark for Positional Detection
[0088] In a particularly preferred mode of the invention, a mark
for positional detection is formed on the substrate. Specifically,
the mark for positional detection is preferably formed on a part
not overlapping with the microarray region for the purpose of
detecting the coordinate position of each spot on the substrate at
the time of spotting the probe or detecting the analyte. It is
particularly effective in such a case where different reagents
should be used according to the spot position on the microarray.
The mark for positional detection may be an optically-readable
mark, an electrically-readable mark, or a magnetically-readable
mark. The optically-readable mark is preferred, because it can
easily be observed using a microscope or an image display
device.
[0089] Examples of the method of forming the mark for positional
detection include: a method of previously forming the mark for
positional detection on a specific position in the substrate by a
printing method, an etching method, a plating method, or the like;
and a method including the processes of simultaneously forming a
latent image of the mark for positional detection in the process of
forming a pattern for arranging patterned regions with at least two
different surface free energies and then applying ink or performing
metal-plating only onto the latent image position.
[0090] The mark for positional detection may be formed on any of
the layers according to the invention as long as its readable
properties are not degraded.
[0091] B. Bio-Microarray
[0092] The inventive bio-microarray is characterized by comprising
the inventive substrate for bio-microarray as described above and
probe biomolecules bonded to the substrate. The inventive
bio-microarray is comprised of the substrate for bio-microarray
having the reflection-suppressing function and thus can relatively
increase the signal intensity of the fluorescent label and can
provide high analytical accuracy. If the inventive bio-microarray
has the specific structure in the anti-reflection layer, it can
have not only the reflection-suppressing function but also the
function of integrating the probe biomolecules at a high density so
that high sensitivity can be provided. If the inventive
bio-microarray is comprised of the substrate for bio-microarray
having the reflection-suppressing function and the reflection
function each formed in a pattern, it can increase the signal
intensity of the fluorescent label and can also suppress the
reflection of unnecessary excitation light so that the resulting
analysis accuracy can further be improved, and thus it can have
various advantages.
[0093] A spotting method comprises a spotting process of forming
spots of a solution containing probe biomolecule on the microarray
substrate and a post-treatment process of immobilizing the
spots.
[0094] The solution (or dispersion) containing probe biomolecule
for use in the spotting process may be prepared in advance or may
be prepared on the substrate by spotting the probe biomolecule and
a solvent, respectively. The probe biomolecule solution may have
any concentration within a normal range. Any solvent may be used
for dissolving the probe biomolecule as long as it is inert to the
biomolecule, and it may appropriately be selected depending on the
use.
[0095] The diameter of the spot formed on the microarray substrate
may vary depending on the use but is preferably in the range from
50 .mu.m.phi. to 400 .mu.m.phi. in terms of quantification
performance and analytical efficiency.
[0096] In the post-treatment process, for example, a treatment for
adjusting the shape of the spots is performed, and then UV
irradiation is performed to crosslink the probe biomolecule so that
the bonding between the molecule and the microarray substrate can
be strengthened.
[0097] The substrate is then immersed in a post-treatment solution
so that the part not loading probe biomolecule is masked. The
post-treatment solution may be appropriately chosen depending on
the use. For example, such a solution may contain succinic
anhydride, N-methylpyrrolidinone, 0.2 M sodium borate (pH 8.0), or
the like. Washing with 95.degree. C. distilled water and 95%
ethanol is then performed to remove an adhering unnecessary
material from the microarray substrate, and centrifugal treatment
and drying are performed so that a microarray is obtained.
[0098] The probe biomolecules may be bonded to the microarray
substrate by the above spotting method or by any other method such
as an inkjet method and a nozzle method.
[0099] As described above, the applicable biomolecule include not
only DNA but also RNA, peptides, proteins, polysaccharides, cells
and tissues.
[0100] The above embodiments are not intended to limit the scope of
the invention. The above embodiments should be taken as an
illustration, and it will be understood that any other products
having a structure substantially equal to that in the technical
scope of Claims and achieving a similar effect should be considered
to be within the scope of the invention as defined in Claims.
EXAMPLES
[0101] The invention is more specifically described with reference
to the examples below.
Example 1
[0102] Forming Anti-Reflection Layer on Substrate
[0103] First, prepared were a solution of 0.4%
polydiallyldimethylammonium chloride (PDDA with a molecular weight
of 400,000 to 500,000 manufactured by Aldrich Chemical Company), a
dispersion of silica particles (MP-1040 manufactured by Nissan
Chemical Industries, Ltd.) and ion exchange water. A commercially
available slide glass was washed and then immersed in the PDDA
solution for two minutes so that PDDA was allowed to adsorb onto
the slide glass. Washing with the ion exchange water was then
performed for two minutes for the purpose of removing unnecessary
PDDA. The PDDA-adsorbing slide glass was then immersed in MP-1040
for one minute for the purpose of forming an anti-reflection layer.
After the silica particles were allowed to adsorb, washing with ion
exchange water whose pH was adjusted to 10 was performed for four
minutes. After the process, an anti-reflection layer comprising
fine particles and having unevenness was formed on both sides of
the slide glass.
[0104] (Forming Immobilization Layer on the Substrate Having
Anti-Reflection Layer)
[0105] The substrate having anti-reflection layer as prepared above
was immersed in a solution of 0.1% poly-L-lysine (manufactured by
Wako Pure Chemical Industries, Ltd.) for five minutes and then
washed with ion exchange water for two minutes. An immobilization
layer comprising the poly-L-lysine was formed at a molecular layer
level by this process. A microarray substrate was obtained by a
series of these processes.
Example 2
[0106] Forming Anti-Reflection Layer on Substrate
[0107] Prepared were a solution of 0.4% polyallyamine hydrochloride
(PAH manufactured by Aldrich Chemical Company), a solution of 0.4%
polyacrylic acid (PAA manufactured by Aldrich Chemical Company),
and ion exchange water. The PAH solution and the PAA solution were
prepared so as to have a pH of 7.8 to 8.5 and a pH of 3.5,
respectively. A commercially available slide glass was washed and
then subjected to 10 cycles of PAH/PAA alternate adsorption (10
cycles of PAH adsorption and 10 cycles of PAA adsorption) so that a
layer-by-layer self-assembled film was prepared. The film was then
treated with acid so as to have a porous structure/uneven structure
for providing an anti-reflection performance. Heat treatment was
then performed to crosslink PAH and PAA by amide bond so that the
anti-reflection structure was fixed. A series of these processes
were according to Hiller et al., Nature Materials, Vol. 1, page 59,
2002.
[0108] (Forming Immobilization Layer on the Substrate Having
Anti-Reflection Layer)
[0109] The substrate having anti-reflection layer was dip-coated
with poly-L-lysine from a solution. An unnecessary solution was
removed by centrifugation so that an immobilization layer was
formed. A microarray substrate was produced by a series of these
processes.
Example 3
[0110] Forming Anti-Reflection Layer on Substrate
[0111] The process of Example 1 was used.
[0112] (Forming Immobilization Layer on the Substrate Having
Anti-Reflection Layer)
[0113] Chemical vapor deposition using 3-aminopropyltriethoxysilane
(manufactured by Across Chemical Co.) was performed to form an
amino group-containing layer on the surfaces of the silica
particles used in the anti-reflection layer. A microarray substrate
was prepared by a series of these processes.
Example 4
[0114] The microarray substrate prepared in Example 3 was
irradiated with vacuum ultraviolet radiation through a quartz
chromium mask having 300 .mu.m.phi. light-blocking parts at a pitch
of 600 .mu.m.phi. so that the organic material was removed from the
part other than the biomolecule-loading part. The part from which
the organic material was removed had a silica-like surface. The
prepared microarray substrate had 300 .mu.m.phi. amino
group-containing regions at a pitch of 600 .mu.m.phi..
Example 5
[0115] The patterned microarray substrate prepared in Example 4 was
used, and octadecyltrimethoxysilane (ODS) was chemically
vapor-deposited thereon. Thereafter, the substrate was thoroughly
washed with cyclohexane so that physically adsorbing ODS was
removed from the amino group-containing pattern. Through a series
of these processes, amicroarray substrate, having array pattern
which contains amino group on the anti-reflection layer and whose
periphery was masked with the octadecyl group, was obtained.
Example 6
[0116] Forming Anti-Reflection Layer on Substrate
[0117] First, prepared were a solution of 0.4%
polydiallyldimethylammonium chloride (PDDA with a molecular weight
of 400,000 to 500,000 manufactured by Aldrich Chemical Company), a
dispersion of polymer particles (AE137 manufactured by JSR
Corporation) and ion exchange water. A commercially available slide
glass was washed and then immersed in the PDDA solution for two
minutes so that PDDA was allowed to adsorb onto the slide glass.
Washing was then performed with the ion exchange water for two
minutes for the purpose of removing unnecessary PDDA. The
PDDA-adsorbing slide glass was then immersed in AE137 for two
minutes for the purpose of forming an anti-reflection layer. After
the polymer particles were allowed to adsorb, washing with ion
exchange water was performed for four minutes. After dried, the
material was heat-treated at 60.degree. C. for 30 minutes so that
adhesion of the particles to the substrate was enhanced. An
anti-reflection layer having unevenness and composed of fine
polymer particles was formed on both sides of the slide glass by a
series of these processes.
[0118] (Forming Immobilization Layer on the Substrate Having
Anti-Reflection Layer)
[0119] The substrate with anti-reflection layer was dip-coated with
poly-L-lysine from a solution. An unnecessary solution was removed
by centrifugation so that an immobilization layer was formed. A
microarray substrate was produced by a series of these
processes.
Example 7
[0120] (Forming Anti-Reflection Layer on Substrate)
[0121] First, prepared were a solution of 0.4%
polydiallyldimethylammonium chloride (PDDA with a molecular weight
of 400,000 to 500,000 manufactured by Aldrich Chemical Company),
two types of dispersions of silica particles (Snowtex 40 and
Snowtex 20L both manufactured by Nissan Chemical Industries, Ltd.)
and ion exchange water. After a commercially available slide glass
was washed, a set of: adsorption of PDDA and washing; adsorption of
Snowtex 40 and washing; adsorption of PDDA and washing; and then
adsorption of Snowtex 20L and washing was performed three times
(the uppermost surface was the silica layer) so that a porous
anti-reflection layer was formed of a laminated structure of the
fine particles using PDDA as an adhesive.
[0122] (Forming Immobilization Layer on the Substrate Having the
Anti-Reflection Layer)
[0123] Chemical vapor deposition using 3-aminopropyltriethoxysilane
(manufactured by Across Chemical Co.) was performed to form an
amino group-containing layer on the surfaces of the silica
particles used in the anti-reflection layer. A microarray substrate
was prepared by a series of these processes.
Example 8
[0124] (Forming Anti-Reflection Layer on the Substrate Having
Light-Absorbing Layer)
[0125] A slide glass having an attached black tape on its back side
was used as a substrate, and an anti-reflection layer was formed
only on the front surface of the substrate by the process according
to Example 1.
[0126] (Forming Immobilization Layer Pattern and Latent Image of
Positional Detection Mark on the Substrate Having the
Light-Absorbing Layer and the Anti-Reflection Layer)
[0127] First, ODS was chemically vapor-deposited to form a layer
containing octadecyl group on the anti-reflection layer. Similarly
to Example 4, vacuum ultraviolet radiation treatment was then
performed using a chromium mask having 200 .mu.m.phi.
non-light-blocking parts at a pitch of 500 .mu.m.phi. (for a
microarray pattern) and a non-light-blocking part in the shape of
50 .mu.m.phi.-wide 2 mm-long lines orthogonally intersecting at the
midpoint (for a positional detection mark pattern).
[0128] (Forming Mark for Positional Detection)
[0129] Microdroplets of silver colloid ink were placed on the
latent image formed by the above process, and then an unnecessary
amount of the ink was sucked up so that the silver colloid ink was
left only on the latent image. It was dried to form a mark for
positional detection, which had a metallic luster.
[0130] (Forming Patterned Immobilization Layer on the Substrate
through the Above Process)
[0131] Chemical vapor deposition of 3-aminopropyltriethoxysilane
was performed on the substrate with the mark for positional
detection, so that 200 .mu.m.phi. amino group-containing
immobilization layers were formed at a pitch of 500 .mu.m.phi. at
the ODS-excluding part on the anti-reflection layer. After a series
of these processes, a microarray substrate was prepared,
comprising: the substrate having the light-absorbing layer on the
back side and the anti-reflection layer on the front side; and the
mark for positional detection and the patterned immobilization
layers formed on the substrate.
* * * * *