JP2816428B2 - Method of patterning functional material and patterned functional material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to immobilization of a functional material.

[0002]

BACKGROUND OF THE INVENTION Techniques for controlling the structure of biological molecules at the surface have a wide range of applications in biosensing, cell guidance, combinatorial chemistry and molecular electronics. Advances in the technology of constructing immobilized molecular structures on surfaces that incorporate biologically active molecules and cells vertically into their structures have resulted in lateral alignment, or patterning, of these structures. There is increasing interest.

[0003]

An object of the present invention is to provide a technique for patterning a plurality of functional materials.

[0004]

Means for Solving the Problems The present invention provides the following items 1 to
Item 7 is provided.

Item 1. A coating film comprising (1) a non-carboxyl group-forming polymer component and (2) a (meth) acrylic acid-tert-butyl ester polymer component is formed on a substrate, and a light or electron beam is applied to the coating film. Irradiating to form a pattern on the substrate surface, applying a functional material having an amino group on the pattern in the presence of a condensing agent, and aligning the functional material along the pattern via an amide bond; A method for immobilizing a conductive material on a substrate.

Item 2. (1) The non-carboxyl group-forming polymer component is polymethyl methacrylate (PMMA),
It consists of polyethyl methacrylate (PEMA), polypropyl methacrylate (PPMA), polymethyl acrylate (PMA), polyethyl acrylate (PEA), polypropyl acrylate (PPA), polymethacrylamide, polyacrylamide, or a copolymer of these monomers. At least one member selected from the group;
And (2) (meth) acrylic acid -tert- butyl ester polymer component, P ^(t Bu) MA (poly -tert- butyl methacrylate), P ^(t Bu) A (poly -tert- butyl acrylate) and ( ^t Bu) MA (tert- butyl methacrylate) and ^(t Bu) a (at least one selected from the group consisting of a copolymer of tert- butyl acrylate)
Item 4. The method according to Item 1, wherein the functional material is a seed.

Item 3. Having a copolymer of (1) a non-carboxyl group-forming monomer and (2) at least one monomer selected from the group consisting of acrylic acid-tert-butyl ester and methacrylic acid-tert-butyl ester on a substrate Forming a coating film, irradiating the coating film with light or electron beam to form a pattern on the substrate surface, applying a functional material having an amino group on the pattern in the presence of a condensing agent, A method for immobilizing a functional material on a substrate, wherein the functional material is aligned along the pattern through the method.

Item 4. Item 4. The method according to any one of Items 1 to 3, wherein the functional material is at least one selected from the group consisting of proteins, enzymes, polysaccharides, nucleic acids, bacteria, viruses, and cultured cells.

Item 5. Item 1 in which the functional material is a nerve cell
A method according to any one of claims 1 to 3.

Item 6. Item 4. A functional material immobilized in a patterned state on a substrate obtained by the method according to any one of Items 1 to 3.

Item 7. Item 7. The functional material according to item 6, wherein two or more types of functional materials are immobilized on a substrate according to different patterns.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is characterized in that a polymer or a copolymer of tert-butyl ester of acrylic acid or methacrylic acid is blended as a component of a coating film, so that the coating film has a photo- or electronic property. When irradiated with a beam, the tert-butyl group of the tert-butyl ester is cleaved to generate a carboxyl group, and the carboxyl group and the amino group of the functional material are
It is characterized in that the functional material is linked via an amide bond by the action of the condensing agent, and as a result, the functional material is aligned only in the portion irradiated with the light or the electron beam.

In the present invention, "non-carboxyl group-forming" means that the compound does not contain a carboxyl group and does not generate a carboxyl group when irradiated with light or an electron beam.

The "non-carboxyl group-forming polymer component" is not particularly limited as long as it is a polymer having no carboxyl group (-COOH) after irradiation with light or an electron beam. For example, polymethyl methacrylate (PMM)
A), polyethyl methacrylate (PEMA), polypropyl methacrylate (PPMA), polymethyl acrylate (PMA), polyethyl acrylate (PE
A), polypropyl acrylate (PPA), polymethacrylamide, polyacrylamide or a copolymer of these monomers, preferably PMMA.

"Non-carboxyl group-forming monomer"
Is not particularly limited as long as it does not form a carboxyl group when irradiated with light or an electron beam after the formation of the copolymer. Examples thereof include methyl methacrylate (MMA),
Ethyl methacrylate (EMA), propyl methacrylate (PMA), methyl acrylate (MA), ethyl acrylate (EA), propyl acrylate (P
A), one or two or more monomers selected from the group consisting of methacrylamide and acrylamide;
Preferably, MMA is used.

In the present invention, (meth) acrylic acid-te
The rt- butyl ester polymer component, P ^(t Bu) MA
(Poly -tert- butyl methacrylate), P ^(t Bu)
A (poly-tert-butyl acrylate) and ( ^tB
u) MA (tert- butyl methacrylate) and ^(t Bu)
Copolymer A (tert-butyl acrylate) and the like, preferably is P ^(t Bu) MA. (Meth) acrylic acid-tert-butyl ester polymer component,
The above-mentioned polymers and copolymers can be used alone or in combination of two or more.

The light- or electron-beam-sensitive coating film of the present invention comprises (1) a non-carboxyl group-forming monomer and (2) tert-butyl acrylate and tert-methacrylic acid.
At least one selected from the group consisting of -butyl ester
It may be composed of a copolymer with a kind of monomer.
Preferred copolymers include methyl methacrylate (M
MA) and tert- butyl methacrylate ^((t Bu) M
And a copolymer with A).

In the coating film, (1) a non-carboxyl group-forming polymer component (first polymer component) and (2) a (meth) acrylic acid-tert-butyl ester polymer component (second polymer component) Is not particularly limited as long as a pattern can be formed by light or electron beam irradiation. For example, first polymer component / second polymer component = 20 to 50/80 to 50% by weight,
Preferably about 50% by weight / about 50% by weight is exemplified as a suitable ratio.

(1) Non-carboxyl group-forming monomer (first monomer) and (2) acrylic acid-tert-butyl ester and methacrylic acid-tert of the copolymer constituting the coating film
At least one selected from the group consisting of -butyl ester
The ratio of the species monomer (second monomer) is not particularly limited as long as a pattern can be formed by light or electron beam irradiation. For example, first monomer / second monomer = 20 to 50
/ 80-50% by weight, preferably about 50% by weight / about 50% by weight
% By weight is exemplified as a suitable ratio.

In the present invention, the carboxyl group formed by irradiation of light or electron beam and the amino group of the functional material may be connected by an amide bond, and is not particularly limited. Preferably, EDC (1-ethyl- 3
-{3-dimethylaminopropyl} -carbodiimide hydrochloride).

In the present invention, the functional material having an amino group is immobilized along a pattern formed on the surface of the substrate. “Align” means immobilizing a functional material at a light or electron beam irradiation site.

The functional material is not particularly limited as long as it is a polymer or macromolecule having an amino group, and examples thereof include proteins, enzymes, polysaccharides, nucleic acids, bacteria, viruses, and cultured cells. Examples include cultured cells such as cells and proteins. Except for the polysaccharide (amino sugar-containing polysaccharide), functional materials other than the above proteins, enzymes, and the like bind via a protein-derived amino group (—NH ₂ ) present on the surface.

The protein is not particularly restricted but includes, for example, hypothalamic hormones (TRH, LHRH, etc.), pituitary hormones (vasopressin, oxytocin, LSH,
TSH, etc.), peptide hormones such as gastrointestinal hormones (secretin, insulin, glucagon), interferons (α, β, γ), interleukins (IL-1 to I-I)
L-6), avidin, albumin, actin, myosin, fibrin, hemoglobin, and other various biologically derived proteins, and these also include glycoproteins.

The enzymes include oxidoreductases (various dehydrogenases, oxidases, reductases, oxygenases, hydroperoxidases), hydrolases (proteases, esterases, glycosidases), transferases (methyltransferase, carboxytransferase, acyltransferase, aminotransferase, Glycosyltransferase), lyase (aldolase, decarboxylase, dehydratase, carboxykinase), isomerase (racemase,
Epimerase, cis-trans isomerase, sugar isomerase, tautomerase, Δ-isomerase, mutase, cycloisomerase) and synthase (DNA ligase, citrate synthase, malate synthase, anthranilate synthase, etc.) are exemplified.

Examples of the polysaccharide include polysaccharides having an amino sugar as a repeating unit, such as glucosamine, mannosamine, and galactosamine.

Examples of the nucleic acid include DNA and RNA.

The bacteria include Escherichia coli, yeast, Bacillus subtilis and the like.

Examples of the virus include influenza virus, herpes virus, adenovirus, AIDS virus, and hepatitis virus (A, B, C).

The cultured cells include nerve cells, tumor cells,
Examples include normal cells such as various organs, organs, tissues, and lymphocytes.

Preferred functional materials include proteins such as avidin and nerve cells.

The material of the substrate on which the pattern is formed on the surface is
Although not particularly limited, for example, glass, silicon, metal,
Ceramics, plastics, natural or synthetic resins and the like can be mentioned. The shape of the substrate is not particularly limited, but is preferably a flat plate.

The patterning of the positive tone following a pattern formation on the substrate surface, the PMMA as a first polymerizable component, as the second polymerizable component P ^(t Bu) M
The case where A is used will be described as an example. PMMA / P (
^t Bu) Instead of MA, a first monomer (eg, PMM
Be a copolymer of A) and a second monomer (e.g., P ^(t Bu) MA), it can be carried out similarly patterned.

The coating film of the light or electron beam sensitivity, PMMA / P ^(t Bu) solvent composed sensitive resin composition from MA (e.g. methyl ethyl ketone, chloroform, toluene, etc.) on a substrate a solution of a used Spin coating, coating, spraying, dipping, etc., and drying,
The sensitive resin is irradiated with light (for example, ultraviolet rays (200 nm or shorter wavelength)) or an electron beam (ビ ー ム 20 kV) to form a pattern.

PMMA and P ( ^t Bu) in the sensitive resin layer
The ratio of MA is not particularly limited as long as capable of forming a pattern, for example, ^{PMMA / P (t Bu) MA} = 20~50 /
80 to 50% by weight; preferably about 50% by weight / about 50% by weight is exemplified as a suitable ratio. This resin composition is composed of the above two commercially available polymers (PMMA and P ( ^t Bu)).
MA) can be used as a mixture,
After mixing the MMA and ^(t Bu) MA which is two monomers, the resulting mixture was refluxed at 100 ° C. to 105 ° C. under acidic conditions (copolymer) can be used.

The molecular weight of PMMA and P ( ^t Bu) MA is
Although not particularly limited, a suitable molecular weight is about 50,000 to 7
5000. These can be easily obtained as commercial products.

A method for forming a pattern is shown in the following scheme.

[0037]

Embedded image

[In the formula, R ₃ represents a portion of the functional material excluding any amino group. m represents an arbitrary integer of 2 or more. The preferable value of m is about 450 to 650.

A culture solution containing a functional material (R ₃ NH ₂ ) or a solvent such as water is added to the substrate. And irradiating the coating layer on the substrate in this state light (e.g. an excimer laser) or electron beam, converts P in the coating layer ^(t Bu) MA (1) poly methacrylic acid (2), the presence of an acid EDC below
Reacts with the amino group of the functional material present in a solvent such as a culture solution or water, whereby the functional material can be aligned (immobilized) along the pattern.

EDC is used in an equimolar or excess amount with respect to the functional material.

The functional material in the case of cells, the substrate 1 cm ² per 10 to 10 ⁷ or so, are 10 to 10 ¹² about fixed when the substrate 1 cm ² per protein.

The specific operation procedure will be described below. scheme
1 and Scheme 2 are merely exemplaryBecause there is,
If necessary, various conditions can be changed as appropriate
You.

Scheme 1 (Preparation of Substrate) (Ii) Starting Material Solid Content = about 5% (PMMA (about 50%) + P ( ^t Bu)
MA (about 50%)) Ratio = about 95% of the solvent, methyl ethyl ketone (I-ii) coating a substrate: silicon, coating the by thickness = 1 mm spin coating PMMA / P ^(t Bu) MA solution, 30 seconds at 500rpm And then at 3000 rpm for 1 minute.

The thickness of the resist film obtained under the above conditions is about 0.60 μm.

(I-iii) Drying step (baking) Apparatus: convection oven Heating condition: 90 ° C. for 30 minutes Scheme 2 (patterning of functional material) (II-i) Patterning energy: 20 kV Printing method: pattern formation by beam scanning (a mask can also be used) (II-ii) Alignment and immobilization of functional material on patterned substrate 1st step: functional material Add solution on patterned resist film Second step: Add EDC solution Third step: Wait 2 hours at room temperature.

(II-iii) Removal of Functional Material Solution First Step: Placing the Substrate on the Bottom of the Well of the Culture Dish Second Step: The resist surface is repeatedly washed.

After the second step, steps (II-i) to (II-iii)
By repeatedly performing the second step, a material in which two or more types of functional materials are aligned and fixed on the substrate is obtained.

[0048]

According to the method of the present invention, any functional material having an amino group can be immobilized along a pattern formed on a substrate.

The pattern formation by light or electron beam irradiation can be performed a plurality of times, and different types of functional materials can be fixed along each pattern. For example, immobilize multiple enzymes with similar primary structures in succession, examine the relationship between enzymes and amino acid sequences, or immobilize multiple neurons in a pattern to form a network-like structure, To study the mechanism of work,
Muscle, blood coagulation system, arrange a plurality of amino group-containing functional materials related to each other such as electron transfer system in a certain positional relationship,
For example, it is possible to check the relationship between the components.

Further, by forming a transistor on a substrate and further immobilizing a nerve cell, it is useful for application as a sensor or an electronic device for sensing a potential change of the nerve cell.

Further, a lattice pattern is formed on the substrate, and a peptide having a different sequence is immobilized on each cell of the lattice pattern for each frame, or an amino acid is arranged in a different sequence on each cell. Sequentially coupled, peptide libraries can be created on a single substrate, providing a convenient way to test which peptides have affinity for which molecules. This indicates that combinatorial chemistry is also useful for application as a screening technique.

[0052]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0053] Example 1 (1) substrate produced PMMA and P ^(t Bu) MA (about 50: about 50 wt%)
Was dissolved in methyl ethyl ketone to prepare a solution having a solids content of about 5%. The solution was placed on a square silicon plate (10 mm × 10 mm, thickness 1.0 mm)
(i) 500 rpm, 30 seconds and (ii) 3000 rpm, 1
Spin coating with a spraying cycle of 1 minute to form a uniform resist film with a thickness of 0.60 μm, and heat convection oven (convect
The substrate was dried at 90 ° C. for 30 minutes in an ion oven.

A pattern was formed on the substrate by using an electron beam irradiation device.

0.5 ml of an aqueous solution obtained by dissolving EDC (4 mg) in 1 ml of water was placed on a substrate placed in a dish-like container.
Was immediately added to the solution, and a solution prepared by dissolving avidin (1 mg) bound to the fluorescent molecule rhodamine in 1 ml of water was immediately added to 0.02%.
Then, the mixture was treated at 23 ° C. for 2 hours to bind avidin to the substrate. The substrate was then washed to remove excess avidin. When the obtained substrate was irradiated with light of 500 nm and observed for fluorescence, it was confirmed that avidin molecules were aligned on the pattern.

The results of the fluorescence test in the same visual field are shown in Reference Photo 1,
It is shown in FIG. Reference photograph 1 is a case observed with a filter suitable for rhodamine, and reference photograph 2 is a case observed with an inappropriate filter. This indicates that fluorescent avidin is bound to the pattern.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C09D 133/06 C09D 133/06 133/26 133/26 C12N 11/00 C12N 11/00 C12Q 1/00 C12Q 1/00 C ( 56) References JP-A-7-168356 (JP, A) JP-A-5-9231 (JP, A) JP-A-4-230758 (JP, A) JP-A-59-48487 (JP, A) Hei 1-39986 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 17/00-17/14 C12N 11/00-11/14 G03F 7/039 G03F 7/38

Claims (7)

(57) [Claims] 1. A coating film comprising (1) a non-carboxyl group-forming polymer component and (2) a (meth) acrylic acid-tert-butyl ester polymer component is formed on a substrate, and said coating film is formed. A pattern on the surface of the substrate by irradiating the functional material having an amino group on the pattern in the presence of a condensing agent, and applying the functional material along the pattern via an amide bond. A method for immobilizing a functional material on a substrate, wherein the functional material is aligned. (1) The non-carboxyl group-forming polymer component is polymethyl methacrylate (PMMA), polyethyl methacrylate (PEMA), polypropyl methacrylate (PPMA), PMA (polymethyl acrylate), polyethyl acrylate. (PEA), polypropyl acrylate (PPA), polymethacrylamide, polyacrylamide or at least one selected from the group consisting of copolymers of these monomers; and
(2) (meth) -tert- butyl ester polymer component acrylic acid, P ^(t Bu) MA (poly-tert-butyl methacrylate), P ^(t Bu) A (poly-tert-butyl acrylate) and ^(t fixed Bu) MA (tert-butyl methacrylate) to ^(t Bu) a (tert- butyl acrylate) copolymer selected from the group consisting of body at least one kind of claim 1, wherein the functional material on the substrate Method. 3. A method according to claim 1, wherein (1) a non-carboxyl group-forming monomer and (2) at least one monomer selected from the group consisting of acrylic acid tert-butyl ester and methacrylic acid tert-butyl ester. Forming a coating film having a copolymer, irradiating the coating film with light or an electron beam to form a pattern on a substrate surface, and forming a functional material having an amino group on the pattern in the presence of a condensing agent. A method for immobilizing a functional material on a substrate, which is applied and aligns the functional material along the pattern via an amide bond. 4. The functional material is a protein, an enzyme, a polysaccharide, a nucleic acid,
The method according to any one of claims 1 to 3, wherein the method is at least one selected from the group consisting of bacteria, viruses, and cultured cells. 5. The functional material is a nerve cell.
The method according to any of the above. 6. A functional material immobilized in a patterned state on a substrate obtainable by the method according to claim 1. 7. The functional material according to claim 6, wherein two or more types of functional materials are immobilized on a substrate according to different patterns.