JP2003035710A - Substrate for biosensor and biosensor using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve the problems that background noise cannot be ignored and it becomes an obstacle in the measuring sensitivity and resolution when a substrate is flat, that a droplet is easy to move when a spot is created, that the position accuracy of the spot is hard to ensure when the substrate is mat-worked to restrain reflection and the direction of emission of fluorescence in a detection is disturbed. SOLUTION: The biosensor has a structure wherein an organic substance layer, a biologically active substance layer and the like are formed sequentially on one face of the substrate 2 on which fine irregularities 3 with a pitch of the wavelength of light or shorter are formed so as to prevent the reflection. Thereby, the problems can be solved.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention can reduce the background of fluorescence measurement caused by the emission of a fluorescent dye used as a label by preventing the reflection of light on the surface of a biosensor substrate, thereby improving sensitivity. The present invention also relates to a biosensor substrate capable of improving resolution, and a biosensor using the same.

[0002]

2. Description of the Related Art A biosensor, for example, is a glass slide-sized substrate for a microscope. For example, various types of gene DNA are attached to individual spots and fixed, and a specimen is labeled with a fluorescent agent or the like in advance. After applying the above, the expression pattern of which DNA portion emitted light was obtained by detecting fluorescence by scanning with a laser beam, and the expression pattern of the gene was obtained by comparing with the known expression pattern. It is used for asking for.

However, a material having a flat surface is used for the substrate of the conventional biosensor, and the presence of the background due to the surface reflection cannot be ignored in the fluorescence measurement, so that the measurement sensitivity is improved and the resolution is improved. It was an obstacle to improvement. Further, there is a drawback that the spot forming liquid is easily moved from a predetermined place when forming a large number of measurement spots. In addition, if the reflection is suppressed by matting the surface, it may be an obstacle to setting a large number of spots with high position accuracy, and may cause, for example, the direction of fluorescence emission to be disturbed during detection.

[0004]

In the present invention, conventionally, when the biosensor substrate is made of a flat material,
The background at the time of measurement due to surface reflection was not negligible, which was an obstacle to improvement of measurement sensitivity and resolution, the point that the spot preparation liquid was easily moved at the time of spot preparation, or matte processing was performed. An object of the present invention is to solve the problem that, when trying to suppress reflection by using a glass plate, it is difficult to secure the positional accuracy in spot placement, or the point that causes the fluorescence emission direction at the time of detection to be disturbed. .

[0005]

Means for Solving the Problems For example, a fine uneven plate in which a fine uneven pattern having a pitch equal to or less than the wavelength of light is formed on the surface of a transparent plate shows the refractive index itself of the transparent plate at the bottom of the unevenness. The closer the surface side is, the lower the proportion of the material that makes up the transparent plate becomes, and instead the higher the proportion of air becomes, so it is as if a number of layers in which the refractive index of light changes continuously are laminated. It is known that it has various effects and can prevent reflection. In the present invention, by bringing in such a special fine unevenness as the structure of the surface of the substrate of the biosensor, it is possible to solve not only the point concerning the reflection of light but also any of the points mentioned as the problems. became.

A first aspect of the present invention relates to a substrate for a biosensor, which has a concavo-convex surface having innumerable fine concavities and convexities formed on one surface of a transparent substrate and having a pitch equal to or less than the wavelength of light. 2nd invention is 1st invention, WHEREIN: The said unevenness | corrugation formed the innumerable minute unevenness | corrugation whose pitch is below the wavelength of light by dividing laser light into 2 or 3 or more, and performing interference exposure. The present invention relates to a biosensor substrate, which is obtained by using an imprinting mold having a mold surface. A third invention relates to the biosensor substrate according to the first or second invention, wherein an organic material layer is laminated on the fine irregularities. A fourth aspect of the present invention relates to the biosensor substrate according to the third aspect, wherein the organic substance layer comprises a silane coupling agent having an amino group or a mercapto group. A fifth invention relates to a biosensor characterized in that a bioactive substance layer is fixed on the organic substance layer of the biosensor substrate of the third or fourth invention. In a sixth aspect based on the fifth aspect, the physiologically active substance layer is a nucleic acid, a non-immunity protein, an immunoglobulin-binding protein, a sugar-binding protein, a sugar that recognizes sugar, a polypeptide having a ligand-binding ability, or The present invention relates to a biosensor characterized by comprising either an oligopeptide or an immune protein. A seventh invention relates to the biosensor according to the sixth invention, wherein the physiologically active substance layer is crosslinked and fixed with an organic substance constituting the organic layer by a crosslinking agent.

[0007]

The present invention will be described with reference to the drawings. FIG. 1 is a diagram showing an outline of the biosensor of the present invention. For example, a minute DNA spot 4 is formed on a substrate 2.
The spots 4 are arranged in a grid pattern, and the number of such spots 4 can be several thousands to several hundreds of thousands, if necessary. Board 2
Shows that the side having the DNA spots 4 is an uneven surface on which innumerable fine unevenness having a pitch equal to or less than the wavelength of light is formed. This fine uneven surface is divided into two or three or more laser beams and subjected to interference exposure, by using an imprinting mold having an uneven mold surface having innumerable fine unevenness with a pitch equal to or shorter than the wavelength of light. It is preferably obtained.

The substrate 2 may be made of any material as long as it is usually used as a biosensor substrate, and is generally glass or an inorganic or organic material transparent to laser light. Consists of. The thickness of the substrate 2 is about 1 mm to 5 mm. If necessary, board 2
May be reinforced on the side or side surface where the DNA spot 4 is not provided. Examples of the organic material that can form the substrate 2 include cellulose diacetate, cellulose triacetate, cellulose acetate butyrate, polyester,
Polyamide, polyimide, polyether sulfone, polysulfone, polypropylene, polymethylpentene,
Polyvinyl chloride, polyvinyl acetal, polyether ketone, polymethylmethacrylate, polycarbonate,
Alternatively, a thermoplastic resin such as polyurethane can be used.

As the shape of the fine unevenness 3 having a pitch equal to or less than the wavelength of light, there may be a cross-sectional shape as illustrated in FIGS. 2 (a) to 2 (e). As the shape of the fine unevenness 3 having a pitch equal to or less than the wavelength of light, in addition to the shape of the upper edge of the cross section shown in FIG. 2 (a) having a sinusoidal shape, the shape of the cross section shown in FIG. The top may be arcuate, the rising portion may be straight, and the shape may be narrower toward the top, or may be triangular as shown in FIG. 2C.

The fine unevenness 3 having the cross-sectional shape shown in FIGS. 2A to 2C is particularly preferable because the depth varies depending on the horizontal position of the fine unevenness 3. As described above, the refractive index of light continuously changes depending on the depth, as shown in FIG.
The upper part has a lower refractive index and the lower part has a higher refractive index.

The shape of the fine irregularities 3 may be a rectangular wave shape as shown in FIG. 2D or an inverted trapezoidal shape as shown in FIG. 2E. Figure 2
In the case of the shape shown in (d), the refractive index is constant in the rectangular wave portion irrespective of the depth, and a constant and predetermined value is realized by determining the pitch and the width of the wave. be able to. Further, in the case of the shape shown in FIG. 2 (e),
In the figure, the upper part has a higher refractive index and the lower part has a lower refractive index.
Since this cross-sectional shape is not an upward concavity, it is difficult to release the mold when using a mold.

The planar arrangement of the fine unevenness 3 is such that
When observed from the upper side of the side with the groove, as shown in a perspective view in FIG.
As shown in the view (concentric circles show contour lines) seen from above in (b) or (c), they may be formed by arranging them side by side in a plane. Both types have antireflection property, but the groove type as shown in FIG. 3 (a) has directionality, and therefore the directionality of reflectance may occur. The two-dimensionally arrayed shape as shown in 3 (b) or (c) is preferable because it does not cause directionality in practice.

Although there are various shapes of the fine irregularities 3a, the pitch (= period) of the waves of the irregularities appearing in the cross-sectional shape is a fine one equal to or less than the wavelength of light. Here, the “wavelength of light” refers to the wavelength of visible light, and thus “pitch equal to or less than the wavelength of light” that represents a fine degree means that the pitch is 400 nm or less. However, it is not limited to 400 nm when the measurement is performed on the longer wavelength side than visible light. The lower limit of the pitch is not particularly limited, but it is preferably 50 nm or more in consideration of accuracy when forming using a mold.

It is preferable that the height difference be 100 nm or more because the larger the difference in wave height of the cross-sectional shape of the fine irregularities 3, the lower the reflectance and the antireflection effect. There is no particular upper limit, but the normal pitch is 50 nm to 400.
Assuming nm, the pitch value is preferably about 100% to 200%, and more preferably about 100 nm to 500 nm.

On the surface of the substrate 2 having the fine irregularities 3, in order to improve the adhesiveness, various treatments that can be usually performed, that is, physical treatments such as corona discharge treatment and oxidation treatment, and anchors are performed. The anchor layer may be formed in advance by performing a chemical treatment by applying an agent (also called a primer agent).

The resin constituting the anchor layer is polyester resin, polyurethane resin, polyethyleneimine resin, polybutadiene resin, acrylic resin, polycarbonate resin, vinyl chloride / vinyl acetate copolymer or the like. One or more of these resins are dissolved in an appropriate solvent to form a paint or ink, which is applied or printed to form the anchor layer. In addition, the anchor layer can be formed by using an alkyl titanate-based anchor agent.

The fine concavities and convexities 3 on the surface of the substrate 2 are most efficiently formed by using an imprinting mold having an inverse shape of the fine concavities and convexities 3 as a mold surface. By pressing the shaping mold in the molten state at the time of plate production, or in the state where the glass plate once produced is melted again,
Can be formed. When the substrate 2 is made of a thermoplastic resin, the fine irregularities 3 can be formed by using a shaping mold in a molten state or a liquid state at the time of manufacturing the substrate 2.

Alternatively, in the case of injection molding of plastic or casting polymerization of acrylic resin, fine irregularities 3 are formed on the inner surface of the mold.
Inject the molten resin in advance, or
By injecting monomer or prepolymer,
The fine unevenness 3 can be formed. Similar results can be obtained by arranging a film-like material having an inverse shape of the fine concavities and convexities 3 on the inner surface of the mold during these injection molding and cast polymerization of the acrylic resin.

The fine irregularities 3 can also be formed by using an ionizing radiation curable resin composition. In this case, the fine unevenness 3 is directly formed on the substrate 2 made of a cured product of the ionizing radiation curable resin composition, and the transparent layer made of the cured product of the ionizing radiation curable resin composition is laminated on the substrate 2. The transparent layer has fine irregularities 3 formed thereon, or the fine irregularities 3 are formed on a thin transparent film different from the substrate 2, for example.
There may be a case where the transparent layer having the layer formed thereon is laminated and a case where the transparent layer is laminated on the substrate 2.

As the ionizing radiation curable resin composition, it is more preferable that the hardness after curing shows a hardness of "H" or more in the pencil hardness test shown in JIS K5400. Also,
The light refractive index of the transparent layer is preferably low in order to exhibit antireflection performance, but for long-term use, surface durability, particularly scratch resistance, is required, and hardness is high. Therefore, it is necessary to increase the density to increase the hardness. Therefore, the refractive index of light of the transparent layer is 1.4 to
1.7, and more preferably 1.6 or less.

The ionizing radiation-curable resin composition is a compound prepared by appropriately mixing a prepolymer, an oligomer and / or a monomer of a compound having a polymerizable unsaturated bond or an epoxy group in the molecule. Specific examples of the compound having a polymerizable unsaturated bond or an epoxy group include polyester (meth) acrylate, urethane (meth) acrylate, epoxy (meth) acrylate, polyether (meth) acrylate, polyol (meth) acrylate. , Melamine (meth) acrylate, triazine acrylate and the like.

Ionizing radiation in the "ionizing radiation-curable resin composition" usually means ultraviolet rays or electron beams. When the ionizing radiation is ultraviolet rays, as the ionizing radiation curable resin composition, acetophenones, benzophenones,
A known photopolymerization initiator or photopolymerization accelerator such as thioxanthone, benzoin, or benzoin methyl ether is added.

The formation of the fine irregularities 3 on the cured product of the ionizing radiation-curable resin composition is efficiently performed by preparing a mold for duplication. In order to prepare a mold for molding, a photosensitive resin is laminated on an appropriate mold base material, and this is exposed by a laser light interference method. In addition, a commercially available photosensitive material for manufacturing a relief hologram may be used by stacking the photosensitive resin on the mold base material.

The exposure is carried out by dividing the laser light into two or three or more and making them interfere with each other. After the exposure, development is carried out according to the kind of the photosensitive resin, and a myriad of fine irregularities having a pitch of not more than the wavelength of the light. A prototype of a mold for forming having a concavo-convex mold surface on which is formed is obtained.

Since the obtained master mold is usually not sufficiently durable, when used for mass production, the master mold is plated with a metal such as nickel and peeled off to form a first metal mold. It is preferable to use as a second metal mold by further plating on the first metal mold to form several second metal molds. In this way, a mold duplicated from the master mold is used. The problem of mold damage and wear can then be avoided.

Forming dies are formed on the roller surface and, if necessary, a stencil plate (multi-faced on the same plate surface), or the shape of the die surface is the length direction of the roller and the circle. The use of endless mold rollers formed continuously in the circumferential direction is suitable for continuous production.

Here, the original mold and the second metal mold have the same shape on the mold surface, and the original mold and the first metal mold have a relationship in which one surface has an opposite mold shape. . In addition, the shape of the fine concavo-convex 3 and the shape of the fine concavo-convex on the mold surface for manufacturing the mold are opposite to each other. Therefore, it is necessary to obtain the fine concavo-convex shape desired for the biosensor substrate. Accordingly, the formation of the metal mold by plating may be repeated to reverse the shape of the fine irregularities. However, when the cross-sectional shape of the fine irregularities is a sine curve, there may be no difference between the original mold shape and the reverse mold shape. In this specification, the shape of the fine irregularities on the mold surface of the imprinting die is, except for the above exceptional cases, the reverse shape so that the desired fine irregularities are formed on the biosensor substrate. It has been formed.

In order to form the fine irregularities 3 on the cured product of the ionizing radiation-curable resin composition, for example, the substrate 2 and the above-mentioned mold for forming are prepared, and first, the both are formed into a liquid ionizing radiation-curable resin. Laminate through the composition. The ionizing radiation curable resin composition may be first laminated on the substrate 2 side, or
You may laminate | stack with a shaping | molding die first.

After lamination, the ionizing radiation curable resin composition is irradiated with ionizing radiation. Upon irradiation with ionizing radiation, the ionizing radiation-curable resin composition is cured to form a cured product and adheres to the substrate 2. Therefore, after irradiation with ionizing radiation, a cured product of the ionizing radiation-curable resin composition is A transparent layer made of a cured product of an ionizing radiation curable resin composition having fine irregularities formed on the surface of the transparent substrate is released on the substrate 2 by releasing the transparent substrate from the imprinting die. Obtainable. By peeling the substrate 2 from the obtained laminate, a transparent layer made of a cured product of an ionizing radiation curable resin composition having fine irregularities formed on the surface can be obtained alone.

In the above process, a continuous transparent film is supplied to the mold roller, an ionizing radiation curable composition is supplied between the mold roller and the transparent film, and the ionizing radiation curable composition is applied together with the transparent film to the mold roller. It can be continuously carried out by irradiating with ionizing radiation in a state of being wrapped around and curing the ionizing radiation-curable composition.

On the fine irregularities 3 of the substrate 2, preferably,
After cleaning the surface with a UV ozone cleaner or the like, a bioactive substance layer is directly formed, or a bioactive substance layer is formed via an organic substance layer to obtain a biosensor.

FIG. 4 is a diagram schematically showing an example of forming a physiologically active substance layer via an organic substance layer. In this example, an amino group is introduced into the side of the substrate 2 having the fine irregularities 3. (FIG. 4 (a)), DNA was immobilized using an amino group (FIG. 4 (b)), and at the time of use, R labeled with a fluorescent agent was used.
It is shown that NA binds to a specific DNA having a complementary structure (FIG. 4 (c)). After that, by applying laser light or the like, fluorescence emission can be obtained from the position where the RNA labeled with the fluorescent agent is bound.

Similarly, a mercapto group is introduced into the side of the substrate 2 having the fine irregularities 3 (FIG. 5 (a)), and a known protein (symbol: Pr) is placed at a fixed position by a crosslinking agent using the mercapto group. If the protein of the sample immobilized on (Fig. 5 (b)) and labeled with a fluorescent agent is of a type that binds to the known protein Pr on the biosensor, it binds (Fig. 4).
(C)) is shown. Again, by applying laser light or the like, fluorescence emission can be obtained from the site where the protein of the sample labeled with the fluorescent agent is bound.

Although the above is a preferred example, the amino group and mercapto group (actually compounds having these) introduced here can be broadly regarded as an organic material layer, and DNA and protein are It can be positioned as a physiologically active substance layer.

The organic material layer preferably has the following (1) to
It is composed of a compound having a functional group such as (8). (1) -COOH, -CHO,, -SH group, -NH ₂
Group, -OH group, = NH group, -CONH ₂ group, -NCO
Group, a —COOR group (R is (CH ₂ ) nH) (n is 1 or more). _{), - (O (CH 2} ) m) nOH group (m,
n is 1 or more), or a compound having any one or more of the functional groups represented by the following "Chemical formula 1".

[0036]

[Chemical 1]

(2) A compound containing nitrogen.

(3) (a) --COOH group and --COOR group (R is (CH ₂ ) nH) (n is 1 or more). ),-
SH group, -NH ₂ group, -OH group, = NH group, -CONH ₂
Group, or - (O (CH ₂₎ m) a compound nOH group functional group alone or a plurality of types of (m, n are both 1 or more as well) are present in total two or more in one molecule, (b) - A copolymer of a compound having two or more NCO groups. In addition, in the following description, (a) and (b) refer to the compounds having the above contents.

(4) A polymer having a plurality of kinds of the compound (a), a plurality of kinds of the compound (ii), or a plurality of kinds of the compound (ii), and a plurality of kinds of the compound (ii) as monomers.

(5) The compound of (a), which is --NH ₂
It has two or more groups and is a —COOH group or a —COOR group (R is (CH ₂ ) nH) (n is 1 or more). ), -SH
Group, -OH group, = NH group, -CONH ₂ group, or -
A polymer having as a monomer a compound having one or more (O (CH ₂ ) m) nOH groups (m and n are both 1 or more).

(6) The compound of (a), which is --NH ₂
Group, -COOH group, -SH group, -OH group, = NH group,-
It has _two or more functional groups of CONH ₂ group or — (O (CH ₂ ) m) nOH group (m and n are each 1 or more) in one molecule, and further has —COOR group (R is (CH ₂ ) nH) (n is 1 or more). ) Is a polymer having as a monomer a compound having one or more of.

(7) The compound of (a), wherein --NH ₂
Group, -OH group or = NH group having one or more functional groups of two or more in one molecule, and further -SH group, -COOH
Group, a —COOR group (R is (CH ₂ ) nH) (n is 1 or more). ), Or a polymer having as a monomer a compound having at least one — (O (CH ₂ ) m) nOH group (m and n are 1 or more).

(8) The compound (b), which is --NCO
Has two or more groups, and a —COOR group (R is (CH ₂ ) n
H) (n is 1 or more). ) Is a polymer having as a monomer a compound having one or more of.

The physiologically active substance constituting the physiologically active substance layer is not particularly limited as long as it interacts with the substance to be measured, and examples thereof include nucleic acid, non-immunity protein, immunoglobulin binding protein, sugar-binding property. Examples thereof include proteins, sugar chains that recognize sugars, polypeptides or oligopeptides having a ligand binding ability, and immunological proteins.

Specifically, the nucleic acids include DNA and RN.
A or PNA (Peptide Nucleic
Acid) and the like, avidin (streptavidin), biotin, or a receptor as a non-immune protein, an immunoglobulin-binding protein such as protein A, protein G, or rheumatoid factor (RF), a lectin such as a sugar-binding protein, Antibodies are examples of immune proteins.

The thickness of the physiologically active substance layer depends on the size of the physiologically active substance itself constituting the physiologically active substance layer, but is 10
It is preferably 0Å to 3000Å, and 100Å to 1
More preferably, it is 000Å.

On the biosensor substrate of the present invention, when the physiologically active substance layer is composed of a physiologically active substance having an amino group, the physiologically active substance having an amino group is combined with the organic substance constituting the organic substance layer. The physiologically active substance layer may be fixed via the organic substance layer by being crosslinked with a crosslinking agent.

The cross-linking agent is not particularly limited as long as it is a water-soluble divalent reagent and can firmly immobilize a physiologically active substance covalently, and examples thereof include glutaraldehyde and periodate. , N, N'-o-phenylenedimaleimide, N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N
-Succinimidyl maleimidoacetic acid, N-succinimidyl-4-maleimidobutyric acid, N-succinimidyl-6-maleimidohexanoic acid, N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1-carboxylic acid, N-
Sulfosuccinimidyl-3-maleimidobenzoic acid, N-
(4-Maleimidobutyryloxy) sulfosuccinimide sodium salt, N- (6-maleimidocaproyloxy) sulfosuccinimide sodium salt, N- (8-
Maleimidocapryloxy) sulfosuccinimide sodium salt, N- (11-maleimidoundecanoyloxy)
Sulfosuccinimide sodium salt or N [2
Examples include-(1-piperazinyl) ethyl] maleimide and dihydrochloric acid, which may be used alone or in combination.

By using such a cross-linking agent, the physiologically active substance constituting the physiologically active substance layer can be firmly fixed by covalent bond, and the fixing of the physiologically active substance can be maintained even if the biosensor is washed. Therefore, an advantage suitable for repeated measurement can be obtained. The cross-linking agent has a layer thickness of 10Å ~
It is preferably 100Å, more preferably 10Å to 50Å.

The biosensor substrate of the present invention is manufactured as follows. First, by the method as described above, the substrate 2 having the fine irregularities 3 having a pitch of not more than the wavelength of light is prepared, and the organic substance layer is laminated on the side having the fine irregularities 3. The formation of the organic material layer is
For example, by causing hydrogen peroxide solution to act on the surface of the substrate 2, OH groups are introduced to the surface of the substrate, and the introduced OH
It can be performed using a group. For example, an amino group or a mercapto group is introduced onto the surface of the substrate 2 using an amino silane coupling agent or a mercapto silane coupling agent.

The organic material layer may be formed by vapor deposition polymerization using the above compound as a monomer,
It can be carried out using a conventional vapor deposition polymerization apparatus. The conditions for vapor deposition polymerization include a film formation rate of 1Å / min to 30
00Å / min, especially 5Å. min-200Å / min
It is preferable to set so that 3000Å / mi
When it exceeds n, it becomes difficult to obtain a uniform vapor-deposited polymerized film.
Specifically, the heating temperature of the monomer raw material is 50 ° C to 150 ° C.
The temperature of the substrate to be vapor-deposited, in this case, a transparent substrate having fine unevenness and having a metal thin film layer laminated thereon, at room temperature,
Alternatively, the pressure is set to 10 ° C. to 20 ° C., and the pressure is 1.0 × 10 ⁻³ Pa.
Is preferred.

A physiologically active substance layer is immobilized on the organic substance layer. Alternatively, the physiologically active substance layer 12 may be fixed to the substrate 2 without the organic substance layer. The immobilization method may be carried out by a conventional method, for example, a predetermined amount of the physiologically active substance can be fixed by contacting the organic substance layer or the substrate 2 for a predetermined time.

When an antibody is used as the physiologically active substance and the Fab fragment of the antibody is immobilized,
The same treatment may be performed after partially degrading the antibody using papain. On the other hand, when the F (ab ′) ₂ fragment of the antibody is immobilized, the same treatment may be performed after partially degrading the antibody with pepsin.

When the physiologically active substance layer is composed of a physiologically active substance having an amino group and is fixed to the organic substance layer, the physiologically active substance having an amino group and the organic substance constituting the organic substance layer are cross-linking agents. By crosslinking with
Can be fixed. Specifically, a cross-linking agent is brought into contact with the organic substance layer to cause a reaction, and after binding the organic substance and the cross-linking agent, a physiologically active substance having an amino group is brought into contact with the organic substance layer to which the cross-linking agent is bound. Then, the cross-linking agent and the amino group are caused to react with each other so as to fix.

On the surface of the substrate 2 having the fine irregularities 3,
When a mercapto group is introduced using a mercapto silane coupling agent, SPDP (SPDP; NS)
uccinimidyl-3- (2-pyridyld
It is preferable to use itio) Propionate) as a crosslinking agent to immobilize proteins and the like.

The biosensor of the present invention uses the substrate unique to the present invention, and attaches and fixes the physiologically active substance by dividing it into individual spots and the like, and the specimen is labeled with a fluorescent agent or the like in advance. After the application, the components in the sample can be confirmed by detecting the fluorescence by scanning with a laser beam. Which part emits light if many kinds of physiologically active substances are applied. The information possessed by the sample can be obtained by obtaining the expression pattern of E. coli and comparing it with a known expression pattern, and as an example, it is highly valuable when used for gene analysis and the like.

[0057]

EXAMPLES Example 1 A photosensitive material formed by applying a photosensitive resin on a plastic film is prepared, and the photosensitive resin is exposed by a laser beam interference method, and then developed. A prototype was obtained in which a myriad of fine irregularities having a pitch equal to or less than the wavelength of light were formed on the mold surface. The mold surface of this prototype was nickel-plated and peeled off to obtain a nickel mold.

The obtained nickel mold was applied so that the mold surface side would contact the surface of the molten glass to obtain a glass substrate having innumerable fine irregularities with a pitch of not more than the wavelength of light. After cleaning the surface of the obtained glass substrate having fine irregularities with an ozone cleaner, the cleaned surface is treated with hydrogen peroxide water to introduce OH groups on the surface, and mercapto-based silane cups are introduced to the introduced OH groups. SH groups were introduced on the surface using a ring agent.

The biosensor substrate thus obtained is, for example, SPDP (N-Succinimid).
yl-3- (2-Pyridyldithio) Pro
abbreviated as "pinate", an SH group- and amino group-reactive cross-linking agent) is used to cross-link the SH group already introduced on the surface with the amino group of the protein, thereby allowing the surface to interact with the substance to be measured. It was possible to immobilize the acting protein and use it as a biosensor for measurement.

In this biosensor, a substrate is imprinted with a concavo-convex surface having innumerable fine concavities and convexities whose pitch is equal to or shorter than the wavelength of light, by subjecting a laser beam to two or three or more and performing interference exposure. Since the protein is immobilized on the surface having fine irregularities obtained by using the mold, there is no surface reflection on the glass plate of the substrate, and the background during fluorescence measurement does not substantially occur, and the irregularities are Since it is a fine one of the wavelength or less, there is no problem such as fluorescence scattering,
There were no problems in terms of measurement sensitivity or resolution. Further, since the unevenness is extremely fine, the dropped liquid does not spread during spotting when immobilizing the protein, and the retention property is good, so that the protein spots could be created accurately.

Example 2 Triacetyl cellulose (=
TAC) film is coated with an acrylate-based UV-curable resin, the nickel-type mold surface obtained in Example 1 is overlaid on the coated surface, and the UV-curable resin is irradiated with UV rays from the TAC film side. Was cured and then peeled off from the mold to obtain a transparent substrate in which a transparent resin layer having innumerable fine irregularities having a pitch of not more than the wavelength of light formed on the surface was laminated on a TAC film. After that, the same procedure as in Example 1 was performed, and similar results were obtained.

[0062]

According to the first aspect of the present invention, since the substrate is provided with fine irregularities having a pitch equal to or less than the wavelength of light, there is no surface reflection and a background is generated during optical measurement. It is possible to provide a substrate for a biosensor that does not have any problem and that the unevenness itself is a fine one that is equal to or less than the wavelength of light, and that the positional accuracy during spotting can be secured. According to the invention of claim 2, the fine concavo-convex is formed by using the imprinting mold having the concavo-convex mold surface formed by the interference exposure with the laser beam. Therefore, the effect of the invention of claim 1 is obtained. It is possible to provide a substrate for a biosensor that can exhibit even more. According to the invention of claim 3, since the organic substance layer is provided on the fine uneven surface, it is possible to provide the biosensor substrate on which the physiologically active substance layer is easily fixed. According to the invention of claim 4, since the organic material layer is composed of a silane coupling agent having an amino group or a mercapto group, it is possible to provide a biosensor substrate on which proteins and the like can be easily fixed. According to the invention of claim 5, since the physiologically active substance layer is fixed on the organic substance layer of the biosensor substrate of the invention of claim 3 or 4, a biosensor that can be directly used for measurement is provided. Can be provided. According to the invention of claim 6, in addition to the effect of the invention of claim 5, a biosensor capable of specifying a substance that constitutes the physiologically active substance layer and using the substance for measurement according to each substance Can be provided. According to the invention of claim 7, in addition to the effect of the invention of claim 6, since the physiologically active substance layer and the organic substance layer are crosslinked and fixed by a crosslinking agent, the physiologically active substance is stably fixed. A biosensor can be provided.

[Brief description of drawings]

FIG. 1 is a diagram showing a biosensor.

FIG. 2 is a diagram showing fine irregularities on a substrate.

FIG. 3 is a diagram showing an array of fine irregularities on a substrate.

FIG. 4 is a diagram illustrating an example of a biosensor.

FIG. 5 is a diagram illustrating another example biosensor.

[Explanation of symbols]

1 biosensor 2 substrates 3 Fine unevenness 4 DNA spot

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // C12M 1/00 C12M 1/00 A C12N 15/09 C12N 15/00 FF term (reference) 2G043 AA01 BA16 CA03 DA02 EA01 GA07 GB05 LA01 MA01 2G054 AA06 BB06 CA21 EA03 GA05 GB02 GE02 GE07 JA10 2G057 AA04 AB04 AC01 BA03 BD04 CB01 DA06 DA09 DA10 4B024 AA11 CA01 CA11 HA12 4B029 AA07 AA21 AA23 BB20 CC08 FA03 FA10 FA10

Claims (7)

[Claims] 1. A substrate for a biosensor, which has an irregular surface on which one side of a transparent substrate is formed with innumerable fine irregularities having a pitch equal to or less than a wavelength of light. 2. The imprinting method, wherein the fine irregularities have a concave-convex mold surface in which innumerable fine irregularities having a pitch equal to or shorter than the wavelength of light are formed by interfering the laser light by dividing the laser light into two or three or more. The biosensor substrate according to claim 1, which is obtained by using a mold. 3. The substrate for biosensor according to claim 1, wherein an organic material layer is laminated on the fine irregularities. 4. The biosensor substrate according to claim 3, wherein the organic material layer comprises a silane coupling agent having an amino group or a mercapto group. 5. A biosensor, wherein a physiologically active substance layer is fixed on the organic substance layer of the biosensor substrate according to claim 3 or 4. 6. The physiologically active substance layer comprises a nucleic acid, a non-immunity protein, an immunoglobulin-binding protein, a sugar-binding protein,
6. The biosensor according to claim 5, comprising a saccharide that recognizes a saccharide, a polypeptide or an oligopeptide having a ligand binding ability, or an immune protein. 7. The biosensor according to claim 6, wherein the physiologically active substance layer is crosslinked and fixed with an organic substance constituting the organic layer by a crosslinking agent.