JP6971034B2 - Analytical equipment - Google Patents

Description

The present invention relates to a laminate, a method for producing the laminate, a surface treatment liquid, a surface treatment method, a biomolecule adsorption layer, and an analytical instrument.

Poly (vinylidene fluoride) (PVDF) membranes are widely used for protein adsorption on Western blots and cytokine adsorption on ELISpot.

Patent Document 1 discloses a method for determining the concentration of a sample using a sample sensor molecule that binds to a porous membrane made of polyvinylidene fluoride.

Japanese Patent Publication No. 2009-545739

In the microarray using the porous membrane made of polyvinylidene fluoride described in Patent Document 1, the amount of protein adsorbed is large, but the transparency of the array itself is low, and a fluorescent substance is used for microscopic observation in a bright field and as a marker. It is difficult to observe if there is.

The present invention has been made in view of the above circumstances, and provides a laminate having high transparency and suitable for fluorescence observation, a method for producing the laminate, and an analytical instrument. The present invention also provides a surface treatment liquid and a surface treatment method which have high transparency and can be used for producing a laminate suitable for fluorescence observation, and a biomolecule adsorption layer.

The first aspect of the present invention is characterized in that a base material and a biomolecule adsorption layer are provided on the base material, and the biomolecule adsorption layer is a poly (vinylidene fluoride) (PVDF) layer. It is a laminated body to be used.

A second aspect of the present invention comprises a step of applying an adhesive to a substrate to form an adhesion layer, and a step of applying a biomolecule adsorption solution to the adhesion layer to form a biomolecule adsorption layer. This is a method for manufacturing the laminated body, which is characterized by the above.

A third aspect of the present invention is a surface treatment liquid comprising a PVDF solution.

A fourth aspect of the present invention is a surface treatment method comprising a step of applying the surface treatment liquid to a substrate.

A fifth aspect of the present invention is a biomolecule adsorption layer comprising a PVDF layer.

A sixth aspect of the present invention is a biomolecule adsorption layer characterized by having a total light transmittance of 90% or more.

A seventh aspect of the present invention is an analytical instrument characterized in that the laminated body is provided on at least a part of the surface.

According to the present invention, it is possible to provide a laminate having high transparency and suitable for fluorescence observation of biomolecules, a method for producing the laminate, and an analytical instrument. Further, according to the present invention, it is possible to provide a surface treatment liquid, a surface treatment method, and a biomolecule adsorption layer which have high transparency and can be used for producing a laminate suitable for fluorescence observation of biomolecules. can.

It is sectional drawing which shows typically one Embodiment of the laminated body of this invention. It is sectional drawing which shows typically the other embodiment of the laminated body of this invention. It is sectional drawing which shows typically one Embodiment of the analysis equipment of this invention.

Hereinafter, embodiments for carrying out the present invention (hereinafter, may be simply referred to as “the present embodiment”) will be described in detail. The present embodiment shown below is an example for explaining the present invention, and is not intended to limit the present invention to the following contents. The present invention can be appropriately modified and carried out within the scope of the gist thereof.

≪Laminated body≫
In one embodiment, the present invention provides a laminate comprising a substrate and a biomolecule adsorption layer on the substrate, wherein the biomolecule adsorption layer is a polyvinylidene fluoride (PVDF) layer. do.

The laminate of the present embodiment has high transparency and can provide analytical equipment and the like suitable for fluorescence observation of biomolecules and the like.

The "biomolecule" in the present specification is, for example, a nucleic acid (naturally-derived) such as DNA, mRNA, miRNA, siRNA, an artificial nucleic acid (for example, LNA (Locked Nucleic Acid), BNA (Bridged Nucleic Acid)). It may be chemically synthesized.); Peptides such as ligands, cytokines, hormones; proteins such as receptors, enzymes, antigens, antibodies; cells, viruses, bacteria, fungi, etc., and proteins. Is preferable.
Samples containing biomolecules include, for example, blood, serum, plasma, urine, puffy coat, saliva, semen, chest exudate, cerebrospinal fluid, tears, sputum, mucus, lymph, ascites, pleural effusion, sheep water, bronchial lavage fluid. , Bronchoalveolar lavage fluid, cell extract, cell culture supernatant and the like.

<Structure>
The structure of the laminated body of the present embodiment will be described in detail below.
FIG. 1 is a cross-sectional view schematically showing an embodiment of the laminated body of the present invention. In FIG. 1, the base material 1 and the biomolecule adsorption layer 2 are laminated in this order.

The laminated body 10 preferably has a total light transmittance of 90% or more, and particularly preferably 93% or more.
Further, the laminate 10 preferably has a haze of 30% or less, more preferably 15% or less, and particularly preferably 5% or less.
The "total light transmittance" (total light transmittance) and "haze" (cloudiness) in the present specification can be measured according to the Japanese Industrial Standards (JIS K7136). By increasing the total light transmittance and further lowering the haze, it is possible to ensure the observability of biomolecules when the laminate of the present embodiment is used.

The laminate 10 shown in FIG. 1 is used, for example, to add a solution containing a biomolecule to the surface of the biomolecule adsorption layer 2 and capture the biomolecule.

FIG. 2 is a cross-sectional view schematically showing another embodiment of the laminated body of the present invention. In FIG. 2, the same components as those shown in the already explained figure are designated by the same reference numerals as in the case of the already explained figure, and detailed description thereof will be omitted.

The laminate 20 shown here is the same as the laminate 10 shown in FIG. 1, except that the laminate 20 is provided with the adhesion layer 3. That is, in the laminated body 20, the base material 1, the adhesion layer 3, and the biomolecule adsorption layer 2 are laminated in this order.

The laminate 20 shown in FIG. 2 is used, for example, to add a solution containing a biomolecule to the surface of the biomolecule adsorption layer 2 and capture the biomolecule.

The laminated body of the present invention is not limited to the one shown in FIGS. 1 and 2, and the structure of a part of the one shown in FIGS. Other configurations may be added to those described so far.

For example, the laminate shown in FIGS. 1 and 2 may have a structure in which the biomolecule adsorption layer is partially laminated on the substrate.
Next, the constituent materials of each layer constituting the laminated body will be described in more detail.

<Constituent material>
(Base material)
The base material is preferably transparent from the viewpoint of observing biomolecules with a fluorescence microscope or the like. Further, in order to enhance transparency, it is preferable not to contain a filler (anti-blocking agent).
As the material of the base material, a transparent low autofluorescent substance is preferable. Preferable examples of the transparent low self-fluorescent material include glass, polyethylene terephthalate, polycarbonate, cycloolefin polymer, polydimethylsiloxane, polystyrene, polyacrylate (acrylic resin) and the like.

More specifically, the polyacrylate (acrylic resin) is, for example, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate). , Poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate), etc. Can be mentioned.

The material constituting the base material may be only one kind, may be two or more kinds, and when there are two or more kinds, the combination and the ratio thereof can be arbitrarily selected.

The base material may be composed of one layer (single layer) or may be composed of two or more layers, and when the base material is composed of a plurality of layers, the constituent materials and the thicknesses of the plurality of layers are the same or different from each other. The combination of these plurality of layers may be not particularly limited as long as the effect of the present invention is not impaired.
In the present specification, not only in the case of a base material, "a plurality of layers may be the same or different from each other" means "all layers may be the same or all layers are different". It may be, and only some of the layers may be the same. ”Furthermore,“ a plurality of layers are different from each other ”means that“ at least one of the constituent materials and the thickness of each layer is different from each other ”. Means.

The thickness of the base material may be, for example, 50 μm or more and 1500 μm or less, 100 μm or more and 1200 μm or less, and for example, 200 μm or more and 1000 μm or less.
Here, the "thickness of the base material" means the thickness of the entire base material, and for example, the thickness of the base material composed of a plurality of layers means the total thickness of all the layers constituting the base material. means.
It is preferable that the base material has a high accuracy of thickness, that is, a base material in which variation in thickness is suppressed regardless of the site.

The base material may contain various known additives such as an antistatic agent, an antioxidant, an organic lubricant, a catalyst, and a softening agent (plasticizer) in addition to the above-mentioned main constituent materials.

In order to improve the adhesion of the base material to other layers such as the biomolecular adsorption layer provided on it, the base material is subjected to sandblasting treatment, unevenness treatment by solvent treatment, corona discharge treatment, electron beam irradiation treatment, plasma. The surface may be subjected to oxidation treatment such as treatment, ozone / ultraviolet irradiation treatment, flame treatment, chromic acid treatment, and hot air treatment.

The base material can be produced by a known method. For example, a base material containing a resin can be produced by molding a resin composition containing the resin.

(Biomolecular adsorption layer)
The biomolecule adsorption layer in the present embodiment is a poly (vinylidene fluoride; PVDF) layer.

The PVDF layer is a polymer of vinylidene fluoride, or a group consisting of vinylidene fluoride and ethylene, propylene, vinyl fluoride, trifluoroethylene, trifluorochloroethylene, tetrafluoroethylene, hexafluoropropylene, and fluoroalkyl vinyl ether. (Hereinafter, it may be referred to as "a monomer copolymerizable with vinylidene fluoride".) It is preferable to contain a copolymer with at least one selected from the above.
When the PVDF layer contains a copolymer of vinylidene fluoride and a monomer copolymerizable with vinylidene fluoride, the copolymer contains vinylidene fluoride in an amount of 80% by mass or more and the vinylidene fluoride. It can be obtained by copolymerizing a monomer containing 20% by mass or less of a monomer copolymerizable with the above.

The PVDF layer is further selected from at least one selected from a copolymer of vinylidene fluoride and a polymerizable unsaturated compound having an acidic group, or a monomer copolymerizable with vinylidene fluoride and the vinylidene fluoride. It may contain a copolymer of a seed and a polymerizable unsaturated compound having an acidic group.

Examples of the polymerizable unsaturated compound having an acidic group include unsaturated dibasic acid having 4 to 8 carbon atoms, methacrylic acid, acrylic acid and the like.

Examples of the unsaturated dibasic acid having 4 to 8 carbon atoms include maleic acid, fumaric acid, citraconic acid, mesaconic acid, 2-pentenedioic acid, methylene succinic acid, allylmalonic acid, isopropyridene succinic acid, 2,4. -Hexadiendioic acid, acetylenedicarboxylic acid and the like can be mentioned.

The material constituting the PVDF layer is at least one selected from a copolymer of vinylidene fluoride and a polymerizable unsaturated compound having an acidic group, or vinylidene fluoride and a monomer copolymerizable with vinylidene fluoride. In the case of a copolymer of a seed and a polymerizable unsaturated compound having an acidic group, the copolymer can be copolymerized with vinylidene fluoride in an amount of 80% by mass or more and, if necessary, vinylidene fluoride. Copolymerization of less than 20% by mass of the above-mentioned monomers, 100 parts by mass of the monomer containing the above, and 0.1 parts by mass or more and 3 parts by mass or less of the polymerizable unsaturated compound having an acidic group. What is obtained is preferable.

A polymerizable unsaturated compound having an acidic group with respect to 100 parts by mass of vinylidene fluoride and, if necessary, at least one monomer selected from the monomer copolymerizable with vinylidene fluoride. The ratio is preferably 0.1% by mass or more and 3% by mass or less, and preferably 0.3% by mass or more and 2% by mass or less. When it is at least the above lower limit value, the effect of introducing an acidic group such as the adhesiveness of the obtained copolymer to the substrate is sufficiently exhibited. On the other hand, when the value is not more than the above upper limit, the chemical resistance of the obtained copolymer is kept high.
Further, for the same reason, it is preferable that the obtained copolymer ^{contains an acidic group of 1 × 10 −5} mol / g or more and 5 × 10 ^-4 mol / g or less.

The material constituting the PVDF layer may be only one kind, may be two or more kinds, and when there are two or more kinds, the combination and the ratio thereof can be arbitrarily selected.

The PVDF layer may be composed of one layer (single layer), may be composed of two or more layers, and when composed of a plurality of layers, the plurality of layers may be the same or different from each other, and these plurality of layers may be formed. The combination of layers is not particularly limited.

The thickness of the PVDF layer is preferably 10 nm or more and 10 μm or less, more preferably 50 nm or more and 5 μm or less, further preferably 100 nm or more and 1 μm or less, and particularly preferably 100 nm or more and 500 nm or less.
When it is at least the above lower limit value, biomolecules can be sufficiently adsorbed, while when it is at least the above upper limit value, high total light transmittance, that is, high transparency can be maintained.

Here, the "thickness of the PVDF layer" means the thickness of the entire PVDF layer, and for example, the thickness of the PVDF layer composed of a plurality of layers means the total thickness of all the layers constituting the PVDF layer. means.
The PVDF layer preferably has a high accuracy in thickness, that is, a layer in which variation in thickness is suppressed regardless of the site.

The method for forming the biomolecule adsorption layer on the base material is not particularly limited, and for example, a method of dropping a predetermined amount of material onto the base material or a contact transfer type coating device such as a roll coater, a reverse coater, or a bar coater is used. Examples thereof include a method and a method using a non-contact coating device such as a spinner (rotary coating device) and a curtain flow coater.

(Adhesion layer)
By using a material having a functional group capable of reacting with an inorganic compound or an organic compound as the constituent material of the adhesion layer, the adhesiveness and adhesion between the base material and the biomolecule adsorption layer can be improved.

The constituent material of the adhesion layer is preferably a base material, or a compound having a functional group capable of reacting with a functional group of a polymer or a copolymer constituting the biomolecule adsorption layer, and is a silane coupling agent. Is more preferable.

Examples of the silane coupling agent include 3-glycidyloxypropyltrimethoxysilane, 3-glycidyloxypropylmethyldiethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-glycidyloxymethyldiethoxysilane, 2-( 3,4-Epoxycyclohexyl) ethyltrimethoxysilane, 3-methacryloyloxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3- (2-aminoethylamino) propyltrimethoxysilane, 3- (2-aminoethyl) Amino) propylmethyldiethoxysilane, 3- (phenylamino) propyltrimethoxysilane, 3-anilinopropyltrimethoxysilane, 3-ureidopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane, 3-mercaptopropylmethyldimethoxy Examples thereof include silane, bis (3-triethoxysilylpropyl) tetrasulfan, methyltrimethoxysilane, methyltriethoxysilane, vinyltrimethoxysilane, vinyltriacetoxysilane, and imidazolesilane.

The material constituting the adhesion layer may be only one kind, may be two or more kinds, and when there are two or more kinds, the combination and the ratio thereof can be arbitrarily selected.

The adhesion layer may be composed of one layer (single layer), may be composed of two or more layers, and when composed of a plurality of layers, the plurality of layers may be the same or different from each other. The combination of layers is not particularly limited.

The thickness of the adhesion layer may be, for example, 1 nm or more and 1 μm or less, and may be, for example, 1 nm or more and 0.5 μm or less.

Here, the "thickness of the adhesion layer" means the thickness of the entire adhesion layer, and for example, the thickness of the adhesion layer composed of a plurality of layers is the total thickness of all the layers constituting the adhesion layer. means.
The adhesion layer preferably has a high accuracy in thickness, that is, a layer in which variation in thickness is suppressed regardless of the site.

The method of forming the adhesion layer on the base material is not particularly limited, and for example, a method of dropping a predetermined amount of material onto the base material, a method of using a contact transfer type coating device such as a roll coater, a reverse coater, or a bar coater, or a method of using a contact transfer type coating device. , A method using a non-contact coating device such as a spinner (rotary coating device) and a curtain flow coater, and the like.

≪Manufacturing method of laminated body≫
In one embodiment, the present invention comprises a step of applying an adhesive to a substrate to form an adhesion layer, and a step of applying a biomolecule adsorption solution to the adhesion layer to form a biomolecule adsorption layer. A method for producing the laminate is provided.

According to the production method of the present embodiment, it is possible to obtain a laminate having high transparency and suitable for fluorescence observation of biomolecules.
Each step of the manufacturing method of this embodiment will be described in detail below.

<Adhesion layer forming process>
First, an adhesive is applied to the substrate. Examples of the adhesive include the same constituent materials as those described in (Adhesion layer) of <Constituent material> of the above-mentioned << Laminated body >>.

The adhesive may contain a solvent. Since the adhesive contains a solvent, the suitability for coating on the surface to be coated is improved.
The solvent is preferably an organic solvent, and examples of the organic solvent include ketones such as methyl ethyl ketone and acetone; esters such as ethyl acetate (carboxylic acid esters); ethers such as tetrahydrofuran and dioxane; cyclohexane, n-hexane and the like. Hydrocarbons; aromatic hydrocarbons such as toluene and xylene; alcohols such as ethanol, 1-propanol and 2-propanol and the like can be mentioned.

Further, as a method for forming the adhesion layer, the same method as the method described in (Adhesion layer) of <Constituent material> of the above-mentioned << Laminated body >> can be mentioned.

When the adhesive contains a solvent, it is preferably dried by heating, and in this case, for example, it is preferably dried at 70 ° C. or higher and 130 ° C. or lower for 10 seconds or longer and 5 minutes or shorter.

<Biomolecule adsorption layer formation step>
Then, the biomolecule adsorption solution is applied to the adhesion layer. Examples of the biomolecule adsorption solution include those obtained by diluting the constituent material described in (biomolecule adsorption layer) of <constituent material> of the above-mentioned << laminate >> with a solvent.
The solvent is preferably an organic solvent, and the organic solvent is, for example, dimethylacetamide (N, N-Dimethyllacetamide; DMA), dimethyl sulfoxide (DMSO), acetonitrile, N-methyl-2-. Examples thereof include an aprotonic polar solvent such as pyrrolidone (NMP).

Further, as a method for forming the biomolecule adsorption layer, the same method as described in (Biomolecule adsorption layer) of <Constituent Material> of the above-mentioned << Laminated Body >> can be mentioned.

Since the formed biomolecule adsorption layer contains a solvent, it is preferably dried by heating. In this case, for example, the temperature is preferably 70 ° C. or higher and 200 ° C. or lower, and 70 ° C. or higher and 130 ° C. or lower. It is more preferable to dry under the conditions. It is preferable to dry under the condition of 10 seconds or more and 5 minutes or less.

≪Surface treatment liquid≫
In one embodiment, the invention provides a surface treatment solution comprising a PVDF solution.

According to the surface treatment liquid of the present embodiment, it is possible to produce a laminate having high transparency and suitable for fluorescence observation of biomolecules.

<PVDF solution>
The PVDF solution is the PVDF-based polymer or copolymer described in (Biomolecule adsorption layer) of <Constituent material> of the above << Laminated product >> and <Biomolecule adsorption of the above-mentioned << Method for producing a laminate >>. It contains the solvent described in the layer forming step>.

The content of the PVDF-based polymer or copolymer contained in the PVDF solution may be any concentration as long as it has high transparency and can form a biomolecule adsorption layer suitable for fluorescence observation of biomolecules, for example, 1 mass. It may be% or more and 25% by mass or less, and may be, for example, 1% by mass or more and 10% by mass or less.

By applying the surface treatment liquid of the present embodiment to a substrate or the like and drying it to form a biomolecule adsorption layer, it is possible to produce a laminate having high transparency and suitable for observing fluorescence of biomolecules. can.

The surface treatment liquid of the present embodiment may further include an adhesive. By providing the adhesive, it is possible to improve the adhesiveness and adhesion between the base material and the biomolecule adsorption layer formed by the PVDF solution.
Examples of the adhesive include the same materials as those described in (Adhesion layer) of <Constituent material> of the above-mentioned << Laminated body >>.

The surface treatment liquid of the present embodiment may further include a solvent for diluting the adhesive. Examples of the solvent include the same solvents as those described in <Adhesion layer forming step> of the above-mentioned << Method for producing a laminated body >>.

≪Surface treatment method≫
In one embodiment, the present invention provides a surface treatment method comprising a step of applying the above-mentioned surface treatment liquid to a substrate.

According to the surface treatment method of the present embodiment, it is possible to produce a laminate having high transparency and suitable for fluorescence observation of biomolecules.

<Applying process>
First, a solution having the same composition as that described in the above-mentioned << surface treatment liquid >> is applied to the substrate.
Examples of the base material to be used include the same as those described in (Base material) of <Constituent material> of the above-mentioned << Laminated body >>.
Further, as a method of applying the surface treatment liquid to the base material, the same method as the method for forming the biomolecule adsorption layer described in (Biomolecule adsorption layer) of <Constituent material> of the above << Laminated body >> can be mentioned. Be done.

The surface treatment method of the present embodiment may further include a drying step after the coating step.
In the drying step, since the biomolecule adsorption layer formed by applying the surface treatment liquid contains a solvent, the biomolecule adsorption layer is heated and dried. In this case, for example, the temperature is preferably 70 ° C. or higher and 200 ° C. or lower, and more preferably 70 ° C. or higher and 130 ° C. or lower. It is preferable to dry under the condition of 10 seconds or more and 5 minutes or less.

Further, in the surface treatment method of the present embodiment, when the surface treatment liquid also includes an adhesive, first, the adhesive diluted to an appropriate concentration with a solvent or the like is applied to the base material or the like and dried by heating to form an adhesion layer. Let me. As the method for forming the adhesion layer and the method for heating and drying, the same method as that described in <Adhesion layer forming step> of the above-mentioned << Method for producing a laminated body >> may be used.
Then, by the above-mentioned <coating step>, the PVDF solution may be applied onto the adhesion layer and dried by heating to form a biomolecule adsorption layer.

≪Biomolecule adsorption layer≫
<First Embodiment>
In one embodiment, the present invention provides a biomolecule adsorption layer composed of a PVDF layer.

According to the biomolecule adsorption layer of the present embodiment, it is possible to obtain a laminate or analytical equipment which has high transparency and is suitable for fluorescence observation of biomolecules.

(PVDF layer)
The biomolecule adsorption layer of this embodiment is composed of a PDVF layer. The PVDF layer in the present embodiment is made of the same constituent material as the (PVDF layer) of the <constituent material> of the above-mentioned << laminated body >>.

The material constituting the PVDF layer may be only one kind, may be two or more kinds, and when there are two or more kinds, the combination and the ratio thereof can be arbitrarily selected.

The PVDF layer may be composed of one layer (single layer), may be composed of two or more layers, and when composed of a plurality of layers, the plurality of layers may be the same or different from each other, and these plurality of layers may be formed. The combination of layers is not particularly limited.

The thickness of the PVDF layer is preferably 10 nm or more and 10 μm or less, more preferably 50 nm or more and 5 μm or less, further preferably 100 nm or more and 1 μm or less, and particularly preferably 100 nm or more and 500 nm or less.
When it is at least the above lower limit value, biomolecules can be sufficiently adsorbed, while when it is at least the above upper limit value, high total light transmittance, that is, high transparency can be maintained.

The PVDF layer preferably has a high accuracy in thickness, that is, a layer in which variation in thickness is suppressed regardless of the site.

The method for forming the PVDF layer is not particularly limited, and for example, a method of dropping a predetermined amount of a material onto a substrate or the like, a method of using a contact transfer type coating device such as a roll coater, a reverse coater, or a bar coater, or a spinner. (Rotary coating device), a method using a non-contact coating device such as a curtain flow coater, and the like can be mentioned.

<Second embodiment>
In one embodiment, the present invention provides a biomolecule adsorption layer having a total light transmittance of 90% or more.

According to the biomolecule adsorption layer of the present embodiment, it is possible to obtain a laminate or analytical equipment which has high transparency and is suitable for fluorescence observation of biomolecules.

The biomolecule adsorption layer of the present embodiment has a total light transmittance of 90% or more, and is particularly preferably 93% or more.
The biomolecule adsorption layer has a haze of 30% or less, preferably 15% or less, and particularly preferably 5% or less.
By increasing the total light transmittance and lowering the haze, it is possible to ensure the observability of biomolecules when the laminate of the present embodiment is used.

The constituent material of the biomolecule adsorption layer of the present embodiment is not particularly limited as long as the total light transmittance is within the above range. Specifically, the same constituent material as the (PVDF layer) of the <constituent material> of the above-mentioned << laminated body >> can be mentioned.

The material constituting the biomolecule adsorption layer may be only one kind, may be two or more kinds, and when there are two or more kinds, the combination and the ratio thereof can be arbitrarily selected.

The biomolecule adsorption layer may be composed of one layer (single layer), may be composed of two or more layers, and when composed of a plurality of layers, these multiple layers may be the same or different from each other. The combination of these multiple layers is not particularly limited.

The thickness of the biomolecule adsorption layer is preferably 10 nm or more and 10 μm or less, more preferably 50 nm or more and 5 μm or less, further preferably 100 nm or more and 1 μm or less, and particularly preferably 100 nm or more and 500 nm or less. preferable.
When it is at least the above lower limit value, biomolecules can be sufficiently adsorbed, while when it is at least the above upper limit value, high total light transmittance, that is, high transparency can be maintained.

The biomolecule adsorption layer preferably has a high accuracy in thickness, that is, a layer in which variation in thickness is suppressed regardless of the site.

The method for forming the biomolecule adsorption layer is not particularly limited, and for example, a method of dropping a predetermined amount of a material onto a substrate or the like, a method of using a contact transfer type coating device such as a roll coater, a reverse coater, or a bar coater, or a method of using a contact transfer type coating device such as a roll coater, a reverse coater, or a bar coater. , A method using a non-contact coating device such as a spinner (rotary coating device) and a curtain flow coater.

≪Analytical equipment≫
In one embodiment, the present invention provides analytical equipment with the laminate on at least a portion of the surface.

The analytical equipment of the present embodiment has high transparency and is suitable for fluorescence observation of biomolecules and the like.
First, the structure of the analytical equipment of the present embodiment will be described in detail below.

<Structure>
FIG. 3 is a cross-sectional view schematically showing an embodiment of the analytical equipment of the present invention. In FIG. 3, the support 5 and the laminate 20 (composed of the base material 1, the adhesion layer 3, and the biomolecule adsorption layer 2) are laminated in this order. Further, the analytical instrument A has a space sandwiched between the laminated body 20 and the top surface member 7 having holes and separated by the side surface member 6. When a sample containing a biomolecule is added, the biomolecule enters the space and is captured by the biomolecule adsorption layer 2.

Further, the analytical instrument A may be provided with the laminated body on at least a part of the surface, and may be provided on the entire surface.

In the analytical equipment A shown in FIG. 3, for example, when a solution containing the biomolecule 4 is added to the surface of the biomolecule adsorption layer 2, the biomolecule 4 is captured and further specifically bound to the biomolecule 4. The biomolecule 4 is detected by adding an antibody 8b to which the substance 8b is bound (that is, a biomolecule-binding antibody 8) and specifically binding the biomolecule 4 to which the biomolecule-binding antibody 8 is captured.

The analytical equipment of the present invention is not limited to the one shown in FIG. 3, and a part of the equipment shown in FIG. 3 has been changed or deleted within the range not impairing the effect of the present invention, or so far. Other configurations may be added to those described.

For example, in the analytical equipment shown in FIG. 3, a laminated body not including the adhesion layer shown in FIG. 1 may be used.
Next, the constituent materials of each layer constituting the analytical equipment will be described in more detail.

<Constituent material>
(Laminated body)
Examples of the laminated body included in the analytical equipment of the present embodiment are the same as those described in the above-mentioned << laminated body >>. In fluorescence observation and the like, since the observation and imaging are mainly performed from the lower surface, the total light transmittance of the laminated body is 90% or more, preferably 93% or more, more preferably 95% or more, and 97. % Or more is more preferable, and 99% or more is particularly preferable.
By increasing the light transmittance, the observability of biomolecules by the analytical equipment of the present embodiment can be sufficiently ensured.

(Support)
The support provided in the analytical instrument of the present embodiment is preferably transparent from the viewpoint of fluorescent observation of biomolecules and the like.
As the material of the support, a transparent low autofluorescent substance is preferable. Preferable examples of the transparent low autofluorescent substance include glass, polyethylene terephthalate, polycarbonate, cycloolefin polymer, polydimethylsiloxane, polystyrene, polyacrylate and the like, and there is no particular limitation.

(Side member)
The side surface member included in the analytical instrument of the present embodiment is preferably transparent from the viewpoint of fluorescent observation of biomolecules and the like.
As the material of the side surface member, the same material as the above-mentioned (support) can be mentioned.

(Top member)
The top surface member included in the analytical instrument of the present embodiment is preferably transparent from the viewpoint of fluorescent observation of biomolecules and the like.
Examples of the material of the top surface member include the same materials as those described above (support).

<Form>
The form of the analytical equipment of the present embodiment is not particularly limited, and examples thereof include cell culture equipment, slides, and microdevices. Examples of the cell culture equipment include a multi-well plate in which an arbitrary number of wells are arranged, a petri dish, and the like. The number of wells includes, for example, 6, 12, 24, 96, 384, 1,536, etc. per plate.

<Others>
The analytical equipment of the present embodiment may further include a buffer used for suspending or dissolving biomolecules, cleaning the analytical equipment, and the like.
Examples of the buffering agent include HEPES-KOH, Tris-HCl, sodium acetate-sodium citrate, sodium citrate-sodium citrate, phosphoric acid, boric acid, MES, PIPESHEEPES-KOH, Tris-HCl, sodium acetate-sodium acetate, and citrate. Acid-sodium citrate, phosphoric acid, boric acid, MES, PIPES and the like, but not limited to these.

Further, the analytical instrument of the present embodiment may further include a reagent for detecting a biomolecule.
The detection reagent is not particularly limited, and may be, for example, an antibody that specifically binds to a target biomolecule.
The antibody that specifically binds to the target biomolecule may be one type or two or more types.
Further, the antibody that specifically binds to the target biomolecule may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of the antibody. The "antibody" here includes all classes and subclasses of immunoglobulin.

As used herein, "specifically binding" means that the antibody binds only to the target protein (antigen), for example, an in vitro assay, preferably a plasmon resonance assay using a purified wild-type antigen. It can be quantified by binding of an antibody to an epitope of an antibody in (for example, BIAcore, GE-Healthcare Uppsala, Swedish, etc.). Binding affinity can be defined by ka (rate constant for antibody binding from antibody-antigen complex), kD (dissociation constant), and KD (kD / ka). Binding affinity, where the antibodies specifically bound to the antigen (KD) is ^preferably not more than 10 -8 mol / ^L, and more to be ^{10 -9} M~10 -13 mol / L preferable.

In the present embodiment, "polyclonal antibody" means an antibody preparation containing different antibodies against different epitopes. That is, when the antibody of the present embodiment is a polyclonal antibody, it may contain a different antibody that contains 40 residues of O-linked GlcNAcized serine and specifically binds to an epitope consisting of a different amino acid sequence.
Further, the "monoclonal antibody" means an antibody (including an antibody fragment) obtained from a substantially uniform population of antibodies.
Also, in contrast to polyclonal antibodies, monoclonal antibodies are meant to recognize a single determinant on an antigen. That is, when the antibody of the present embodiment is a monoclonal antibody, it is an antibody isolated from a component of the natural environment.

In the present embodiment, the "functional fragment" of an antibody means a part (partial fragment) of the antibody that specifically recognizes a target protein. Specifically, Fab, Fab', F (ab') 2, variable region fragment (Fv), disulfide bond Fv, single-chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And polymers of these.

In addition, the labeling substance may be bound to the antibody that specifically binds to the target biomolecule.
Examples of the labeling substance for the antibody include stable isotopes, radioisotopes, fluorescent substances, enzymes, magnetic substances and the like.

Examples of stable isotopes include, but are not limited to, ¹³ C, ¹⁵ N, ² H, ¹⁷ O, and ^{18 O.}
Radioisotopes, such as ^{3 H, 14 C, 13 N} , 32 P, 33 P, 35 S and the like, but are not limited to.
Examples of the fluorescent substance include, and are not limited to, cyanine dyes (for example, Cy3, Cy5, etc.), rhodamine 6G reagents, and other known fluorescent dyes (for example, GFP, FITC (Fluorescein), TAMRA, etc.).

Examples of the enzyme include alkaline phosphatase and peroxidase (HRP).
When the labeling substance is an enzyme, it is preferable to use an enzyme substrate. As the enzyme substrate, in the case of alkaline phosphatase, p-nitrophenyl phosphate (pNPP), 4-methylumbelliferyl phosphate (4-MUP) and the like can be used, and when the enzyme is peroxidase. , 3,3'-diaminobenzidine (DAB), 3,3', 5,5'-tetramethylbenzidine (TMB), o-phenyllenediamine (OPD), 2,2-azinodi- (3-ethylbenzothiazolin-6-) Sulfonic acid) (ABTS), 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) and the like can be used.

Examples of the magnetic material include gadolinium, Gd-DTPA, Gd-DTPA-BMA, Gd-HP-DO3A, iodine, iron, iron oxide, chromium, manganese, or a complex thereof, or a chelate complex, and the like. Not done.

Further, the detection reagent includes, in addition to the antibody that specifically binds to the target biomolecule, a secondary antibody in which the labeling substance is bound to the antibody against the antibody that specifically binds to the target biomolecule. May be.
When a secondary antibody is prepared by binding an antibody to an antibody that specifically binds to the target biomolecule, an ELISA method using an antibody measurement system, an indirect fluorescent antibody method, a CLEIA method using an antibody measurement system, or the like is used. Therefore, the target biomolecule can be detected and quantified.

An animal (eg, mouse, rat, hamster, rabbit, etc.) from which the antibody specifically binds to the biomolecule of interest is bound to an antibody against the antibody of interest. It is preferably an antibody against monkeys, goats, etc.). For example, when the origin of the antibody that specifically binds to the target biomolecule is mouse, it is preferably an anti-mouse antibody. In addition, all classes and subclasses of immunoglobulins are included in those in which the labeling substance is bound to the antibody against the antibody that specifically binds to the biomolecule of interest.
Examples of the labeling substance include those similar to those exemplified in the above-mentioned explanation of "antibody that specifically binds to the biomolecule of interest".

<Use>
The analytical equipment of the present embodiment can be used, for example, for screening a drug effective for a target disease.
Further, the analytical equipment of the present embodiment can be used for culturing adhesive cells or the like because the biomolecule adsorption layer has a property of adsorbing a membrane protein or the like on the cell surface.
The analytical instrument of the present embodiment is, for example, a protein using an antigen-antibody reaction such as RIA method, EIA method (including ECLIA method and ELISA method), CIA method (including CLIA method and CLIIA method), and FIA method. It can be used for detection and quantitative systems.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples and the like.

[Production Example 1] Production of laminate 1 a to 1 e (1) Preparation of PVDF solution First, poly (vinylidene fluoride) (PVDF, molecular weight 2.8 × ¹⁰ 5) and (Aldrich Co.) 3.3,5. Five different concentrations of PVDF solutions were prepared using N-methyl-2-pyrrolidone (NMP) to be 0, 6.7, 10, and 14.0% by weight.

(2) Formation of PVDF layer Next, a PVDF solution of each concentration was spin-coated on a glass substrate (Eagle-XG (registered trademark), 0.7 mm, manufactured by Corning Inc.) (condition: rotation speed should be the specified film thickness). Setting, 20 seconds). Then, it was heated at 120 ° C. for 1 minute to form a PVDF layer, and laminated bodies 1a to 1e having different film thicknesses were produced. The film thickness of each laminate is shown in Table 1 below.

(3) Measurement of total light transmittance and haze value Next, for the laminates 1a to 1e produced in (2), a haze meter (HZ-2A manufactured by Suga Test Instruments Co., Ltd.) and D65 as a light source were used. The total light transmittance and the haze value were measured according to the method of JIS K7136. The results are shown in Table 1 below.

(4) Cell observation Next, the laminates 1a to 1e produced in (2) were stained with calcein-AM of namalva cells (lymphoblast cell line) on the upper surface (the surface on which the PVDF layer was formed). ), And microscopic observation was performed from the lower surface (glass substrate). As a control, cells were dropped onto a PVDF porous membrane (manufactured by Merck Millipore, film thickness 130 μm), and microscopic observation was performed from the lower surface. The results are shown in Table 1 below.
The viewpoints of evaluation in cell observation were as follows.
○: No effect on cell observation with a microscope △: Concavities and convexities on the substrate are observed ×: Cell observation with a microscope is not possible

[Production Example 2] Production of laminates 2a to 2e (1) Preparation of PVDF solution Instead of PVDF (manufactured by Aldrich), poly (vinylidene fluoride-co-hexafluoropropylene) (PVDF / HFP, molecular weight 4.6) × 10 ⁵ ) (manufactured by Aldrich) was used, but the same method as in (1) of Production Example 1 was used to prepare PVDF solutions having five different concentrations.

(2) Formation of PVDF Layer Next, using the same method as in (2) of Production Example 1, laminates 2a to 2e having different film thicknesses were produced. The film thickness of each laminate is shown in Table 1 below.

(3) Measurement of total light transmittance and haze value Next, for the laminates 2a to 2e produced in (2), the total light transmittance and haze value are used in the same manner as in (3) of Production Example 1. Was measured. The results are shown in Table 1 below.

[Production Example 3] Production of Laminates 3a to 3e (1) Preparation of PVDF Solution Instead of PVDF (manufactured by Aldrich), KF polymer # 9130 (carboxylic acid-modified PVDF, molecular weight 2.8 × 10 ⁵ ) (Kureha) A PVDF solution having five different concentrations was prepared using the same method as in (1) of Production Example 1 except that the above was used.

(2) Formation of PVDF Layer Next, using the same method as in (2) of Production Example 1, laminates 3a to 3e having different film thicknesses were produced. The film thickness of each laminate is shown in Table 1 below.

(3) Measurement of total light transmittance and haze value Next, for the laminates 3a to 3e produced in (2), the total light transmittance and haze value are used in the same manner as in (3) of Production Example 1. Was measured. The results are shown in Table 1 below.

From Table 1, in the laminate using any of PVDF, PVDF / HFP, and carboxylic acid-modified PVDF, the total light transmittance exceeded 90%. In addition, the haze value tended to increase as the film thickness increased.
In addition, the haze value tended to be lower in the laminate using PVDF / HFP and the carboxylic acid-modified PVDF as compared with the laminate using PVDF.

[Test Example 1] Protein adsorption test (1) Preparation of laminate A protein adsorption test was conducted using the laminates 1a, 2a, and 3a having a film thickness of 0.2 μm produced in Production Examples 1 to 3. Further, as a control, a glass substrate (Eagle-XG (registered trademark), 0.7 mm, manufactured by Corning Inc.) was used.

(2) Protein adsorption Next, 10 μL of 20 μg / mL Alexa488-labeled goat anti-mouse IgG antibody (# A11001 manufactured by Life Technologies) was added dropwise to each laminate and glass substrate, and the mixture was allowed to stand for 1 hour. Then, it was washed with PBS (−) three times and then with pure water. Next, the amount of adsorbed protein was measured for fluorescence intensity with Typhoon TRIO (GE Healthcare Japan Co., Ltd., excitation light 488 nm, filter 520 nm). Next, the autofluorescence intensity of each laminate, glass substrate, or polystyrene dish was subtracted from each fluorescence intensity, and the value was used to calculate the relative intensity with the glass substrate. The results are shown in Table 2 below.

From Table 2, high fluorescence intensity was observed in each of the laminates. Further, as compared with the laminate using PVDF, the amount of protein adsorbed tends to decrease in the laminate using PVDF / HFP, and the amount of protein adsorbed tends to increase in the laminate using carboxylic acid-modified PVDF. Was done.

According to the present invention, it is possible to provide a laminate having high transparency and suitable for fluorescence observation of biomolecules, a method for producing the laminate, and an analytical instrument. Further, according to the present invention, it is possible to provide a surface treatment liquid and a biomolecule adsorption layer which have high transparency and can be used for producing a laminate suitable for fluorescence observation of biomolecules.

1 ... Substrate, 2 ... Biomolecule adsorption layer, 3 ... Adhesion layer, 4 ... Biomolecule, 5 ... Support, 6 ... Side member, 7 ... Top surface member with pores, 8 ... Fluorescent substance binding antibody, 8a ... Antibodies, 8b ... Fluorescent substances, 10, 20 ... Laminates, A ... Analytical equipment.

Claims (6)

It is used for fluorescent observation of biomolecules.
An analytical instrument in which a laminate having a biomolecule adsorption layer provided on a substrate is provided on a support, and the support, the substrate, and the biomolecule adsorption layer are laminated in this order.
The thickness of the biomolecule adsorption layer is 10 nm or more and 5 μm or less, and the thickness is 10 nm or more and 5 μm or less.
The total light transmittance of the laminated body is 90% or more, and the total light transmittance is 90% or more.
Wherein the biomolecule adsorbent layer, poly (vinylidene fluoride) (PVDF) Sodea is,
Analytical equipment in which biomolecules are captured by the biomolecule adsorption layer when a sample containing biomolecules is added to the surface of the biomolecule adsorption layer . The PVDF layer is a polymer of vinylidene fluoride or a group consisting of vinylidene fluoride and ethylene, propylene, vinyl fluoride, trifluoroethylene, trifluorochloroethylene, tetrafluoroethylene, hexafluoropropylene, and fluoroalkyl vinyl ether. The analytical equipment according to claim 1, which comprises a copolymer with at least one selected from the above. The PVDF layer is further formed by a copolymer of vinylidene fluoride and a polymerizable unsaturated compound having an acidic group, or vinylidene fluoride and ethylene, propylene, vinyl fluoride, trifluoroethylene, trifluorochloroethylene, and the like. tetrafluoroethylene, hexafluoropropylene and at least one and comprises a copolymer of a polymerizable unsaturated compound having an acidic group, analytical equipment according to claim 2 selected from the group consisting of fluoroalkyl vinyl ether. Polymerizable unsaturated compound having the acid group, an unsaturated dibasic acid having 4 to 8 carbon atoms, methacrylic acid, and is at least one selected from the group consisting of acrylic acid, for analysis according to claim 3 Equipment . With an adhesion layer between the substrate and the biomolecule adsorption layer, analytical equipment according to any one of claims 1-4. The analytical instrument according to any one of claims 1 to 5, which is used for screening a drug effective for a target disease.

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