JP2003161739A - Substrate for microchip, and microchip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DNA chip substrate that compensates for the weak point of glass used as a substrate for DNA chips, has high surface accuracy, especially flatness, and eliminates the need for surface treatment for immobilization. <P>SOLUTION: A varnish comprising an epoxy resin (a), an epoxy resin (b) containing hydroxyl group and epoxy group at the end, and a curing agent is dipped into a base material to create a prepreg. One or a plurality of prepregs are overlapped as one set and are heated and pressurized to compose the substrate for DNA chips. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a DNA chip substrate and a DNA chip constructed using the substrate.

[0002]

2. Description of the Related Art As a substrate of a DNA chip used for gene analysis and the like, glass represented by a slide glass has been conventionally used. The reason is that glass can realize extremely high surface precision, especially flatness.
That is, a solution containing a DNA fragment arrayed on a DNA chip and a sample (DNA) to be examined labeled with a fluorescent substance is poured on the chip to determine which DNA on the chip has hybridized. It is read and detected by a focus laser scanner, but at this time it is necessary to focus on an extremely minute position, and even if there are slight irregularities on the surface, there will be a deviation from the focus that was initially set. Is extremely strict, and is required to be several microns or less.

However, since such a surface precision cannot be obtained by the ordinary plate glass manufacturing method, at present, by applying a considerable number of man-hours and polishing to a level similar to that of optical glass, these precision and flatness are obtained. Has been realized. Furthermore, although it is necessary to immobilize the DNA fragment on the glass surface, it cannot be immobilized as it is, and therefore surface treatment is required. However, the surface treatment of glass is difficult, and post-processes such as coupling treatment are unavoidable, complicating the manufacturing process. Furthermore, there is a problem of reliability that the glass is liable to be broken due to falling or the like. In addition to the above technical difficulties in handling, the DNA chip has a cost problem due to the price of the polishing glass, and an inexpensive and simple DNA chip substrate and a DNA chip have been desired.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a DNA chip more simply by compensating for the drawbacks of glass used as a DNA chip substrate.

[0005]

Means for Solving the Problems The present inventor says that when a substrate is prepared using a specific epoxy resin raw material, DNA is immobilized on the substrate as compared with the case where another epoxy resin raw material is used. Focusing on the phenomenon, it was found that the α-glycol group-containing epoxy resin contained as an impurity in the specific raw material plays an important role. The present inventor has completed the present invention as a result of extensive studies based on this finding.

That is, the present invention provides (1) an epoxy resin (a) represented by the formula 1 and an epoxy resin (b) represented by the formula 2.
And a substrate for a microchip that is composed of at least a curing agent,

[0007]

[Chemical 3] (M = 0 or more integer)

[0008]

[Chemical 4] (N is an integer of 0 or more, R is a hydrocarbon residue having 1 to 10 carbon atoms (may include a functional group such as alcoholic OH)).

(2) The microchip substrate according to (1), wherein the content of the epoxy resin (b) is 2 to 20 gram equivalents relative to 100 gram equivalents of the epoxy resin (a),
(3) A microchip using the microchip substrate according to (1) or (2).

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The DNA chip as used in the present invention has a broad meaning including a microarray. (Epoxy Resin (a)) The epoxy resin (a) used in the present invention can be easily obtained by reacting bisphenol A with epichlorohydrin. As the degree of polymerization increases (as m in the formula increases), the form changes to liquid, semi-solid, and solid, but there is no particular limitation in use.
However, m is 0 to 40 from the viewpoint of easy availability.
Resins up to and particularly preferably resins from 0 to 20 are generally used. It is also possible to use two or more kinds of epoxy resin mixtures having different degrees of polymerization as the resin (a). When a commercially available epoxy resin (a) is used, the epoxy resin mixture has different degrees of polymerization,
It is customary to indicate the properties by the number average molecular weight, the weight average molecular weight, the average degree of polymerization or the epoxy equivalent.

(Epoxy Resin (b)) Next, the epoxy resin (b) containing a hydroxyl group and an epoxy group at each of the terminals used in the present invention will be described in detail. In the present invention, the epoxy resin (b) plays the most important role in immobilizing DNA and the like. Morphological confirmation has not yet been taken, but in view of the molecular structure, the epoxy resin (b) is grafted from the surface of the substrate via the epoxy group, and the epoxy resin (b) is linked to the target via the terminal OH in the free space on the opposite side. It is presumed that it has an excellent DNA-immobilizing ability because it can bind to the DNA etc.

The epoxy resin (b) can be produced, for example, by adding a primary alcohol such as methanol to one end of the bisphenol A type epoxy resin. Although there is no particular limitation on the degree of polymerization, it can be used,
From the viewpoint of easy availability, n is generally 0 to 40, particularly preferably 0 to 20. The number of carbon atoms in the hydrocarbon residue is limited to 10 or less not only for ease of synthesis, but when the number of carbon atoms is greater than 10, the compatibility with the epoxy resin (a) becomes poor and steric hindrance becomes large. Or target DNA
This is because the immobilization process such as the above will be hindered. Also, α-
An OH group may be present in addition to the terminal, such as a glycol group. It is also possible to use, as the epoxy resin (b), an epoxy resin mixture having two or more kinds of different polymerization degrees or different hydrocarbon residues, each containing a hydroxyl group and an epoxy group at the terminal.

In the present invention, from the viewpoint of immobilizing DNA or RNA, it is desirable that the concentration of the epoxy resin (b) is high in order to increase the number of OH groups grafted from the surface of the substrate, but dimensional stability as a DNA chip substrate. From the viewpoints of properties, flatness, and rigidity, it is preferable that the concentration is low. That is, the content of the epoxy resin (b) is preferably 2 to 20 gram equivalents, and more preferably 5 to 15 gram equivalents, relative to 100 gram equivalents of the epoxy resin (a). Two
If it is less than gram equivalent, immobilization tends to be insufficient,
On the other hand, if it exceeds 20 gram equivalents, the dimensional stability and rigidity of the substrate tend to be insufficient.

(Curing Agent) As the curing agent, amine-based, acid-anhydride-based, polyamide-based, etc., which are commonly used for epoxy resins, can be used. In particular, an acid anhydride type curing agent having a long pot life and low toxicity is preferably used. Examples of acid anhydride hardeners include phthalic anhydride, hexahydrophthalic anhydride, hexamethylnadic acid, pyromet acid anhydride and dodecenyl succinic anhydride. At this time, a curing accelerator such as a tertiary amine or imidazole may be used together.

(Filler, solvent, etc.) Coupling, if necessary, for the purpose of improving the fixability with an inorganic filler such as aluminum hydroxide, silica, talc, wollastonite, magnesium hydroxide, clay, etc. or a substrate. It is also possible to add agents or pigments such as carbon black to the composition.

In the present invention, a varnish is used in the form of an epoxy resin (a), an epoxy resin (b) and a curing agent, but a solvent is often used. The solvent must have good solubility in a part or the whole of the composition, but a poor solvent can be used as long as it does not adversely affect the composition. Examples of such solvents include acetone, methyl ethyl ketone, methyl isobutyl ketone, ketone solvents such as cyclohexanone, toluene, xylene, aromatic hydrocarbon solvents such as mesitylene, methyl cellosolve, ethyl cellosolve, butyl cellosolve, isobutyl. Cellsolve, diethylene glycol monomethyl ether, triethylene glycol monomethyl ether, propylene glycol monomethyl ether, dipropylene glycol monomethyl ether, propylene glycol monopropyl ether, dipropylene glycol monopropyl ether, ethylene glycol monoisopropyl ether, diethylene glycol monoisopropyl ether, diethylene glycol mono Various glycol ether solvents such as butyl ether, methyl ether Ester solvent such as sorbacetate, ethyl cell solvate acetate, butyl cell solvate acetate, ethyl acetate, dialkyl glycol ether type solvent such as ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, diethyl glycol dibutyl ether, N, N-dimethylacetamide , N, N-dimethylformamide, N-methyl-
There are amide-based solvents such as 2-pyrrolidone and alcohol-based solvents such as methanol and ethanol, and these may be used in combination of several kinds.

(Substrate and Manufacturing Process) The substrate of the present invention is not particularly limited, and may be glass woven fabric, glass non-woven fabric,
Alternatively, a cloth or the like having a component other than paper and glass is used. D
From the viewpoint of dimensional stability and rigidity of the NA chip substrate, glass woven cloth and glass nonwoven cloth are preferably used. A prepreg for a DNA chip substrate can be obtained by coating and impregnating the above-mentioned base material with a varnish and drying in a drying oven within a range of 20 to 200 ° C. Here, a plurality of prepregs each having a different composition of the epoxy resin (a), the epoxy resin (b), the curing agent or the base material are overlapped,
A laminated board obtained by heating and pressurizing under the above conditions can also be used as a DNA chip substrate.

The prepreg obtained in the above steps has a temperature range of 100 ° C. to 200 ° C., a pressure range of 0 to 30 MPa, preferably 10 MPa or less, and more preferably 5 MPa.
A product for a DNA chip substrate can be manufactured by applying a pressure of MPa or less. Particularly in applications where dimensional stability is required, in addition to the method of molding at low pressure, a method of taking strain by reducing the cooling rate or reheating without molding after molding can be generally adopted.

[0019]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. First, the evaluation method in the present invention is shown below. [Example 1] First, in making a varnish, its composition by weight is as follows. Composition of varnish (1) Epoxy resin (a) 100 parts by weight (number average molecular weight Mn900 → average degree of polymerization n = 2.
0) (2) 7 parts by weight of an epoxy resin containing a hydroxyl group and an epoxy group at each end as shown in Formula 3

[0020]

[Chemical 5]

(3) Hardener: Hexahydrophthalic anhydride 50 parts by weight (4) Solvent: Acetone 50 parts by weight The components (1) to (4) were mixed to prepare a uniform varnish. Next, the varnish was applied to a glass woven fabric having a thickness of 0.18 mm (mass: 205 g / m2) with a resin content of 42 to 45.
The glass woven fabric prepreg was obtained by impregnating and drying the prepreg so that the amount of the prepreg was glass. Six glass woven prepregs were superposed and compression-molded at a molding temperature of 150 ° C. and a pressure of 3 MPa for 90 minutes to obtain a rectangular substrate of L770 × W270 × t1 mm. From this substrate, slide glass molding (dimension L76
100 mm × W26 mm × t1 mm) can be collected.

Example 2 In the varnish of Example 1, 30 parts by weight of silica (manufactured by Takimori, Crystallite VX-3), 100 parts by weight of the resin component in the varnish, aluminum hydroxide (Showa Light Metal Heidi Co., Ltd.) were used. 70 parts by weight of Light H-42) was added to prepare a varnish containing an inorganic filler. Inorganic filler-containing varnish made of non-woven glass (made by Nippon Vilene, EP
-4075) was impregnated and dried so that the contents of the resin, the curing agent and the inorganic filler were 90 parts by weight to obtain a glass nonwoven fabric prepreg. Next, the glass non-woven fabric prepreg was used as an intermediate layer, the prepreg used in Example 1 was placed on the surface layer (both upper and lower sides), and the molding temperature was 150 ° C. and the pressure was 3 MPa.
After compression molding for 0 minutes, a rectangular substrate of L770 × W270 × t1 mm was obtained.

[Embodiment 3] L7 is manufactured in the same manner as in Embodiment 1.
A rectangular substrate of 70 × W270 × t1 mm was obtained. However, a varnish was prepared by using the following components instead of the varnish of Example 1. Composition of varnish (1) Epoxy resin (a) 100 parts by weight (average molecular weight Mn1600 → average degree of polymerization n = 4.
4) (2) 12 parts by weight of an epoxy resin containing a hydroxyl group and an epoxy group at each end as shown in Formula 4

[0024]

[Chemical 6]

(3) Curing agent: Hexamethyl nadic acid 70 parts by weight (4) Facial agent: Carbon black 3 parts by weight (5) Solvent: Methyl ethyl ketone 50 parts by weight [Comparative Example 1] Terminal as shown by formula (3) To each without addition of an epoxy resin containing a hydroxyl group and an epoxy group,
A varnish was prepared by the same production method as in Example 1, and a glass woven fabric prepreg was obtained using the varnish. Six pieces of the glass woven prepregs were superposed, and compression molded at a molding temperature of 150 ° C. and a pressure of 3 MPa for 90 minutes to obtain L770 × W2.
A 70 × t1 mm rectangular substrate was obtained.

[Comparative Example 2] White glass (Dimension: L76m)
m × W26 mm × t1 mm) was used to carry out spot DNA substrate immobilization and reliability evaluation described by the following evaluation method. Next, the evaluation method in the present invention is shown below.

(2) Flatness: L770 × W270 × t1 mm which is a size slightly more than 10 times as large as a slide glass molded product.
The warpage of the rectangular substrate was measured, and the value was described in the table. If it was within 50 microns, it was marked with ◯, and if it exceeded 50 microns, it was marked with x. However, in the case of the slide glass of Comparative Example 2, L76 mm x W26 mm x t1 mm
The warp was measured and confirmed to be 5 microns.

(2) DNA immobilization efficiency: The slide glass-like molded article was used as a substrate and evaluated according to the following protocol. (Preparation of aminated oligo DNA) 5'-TAGAAGC
Oligo DNA (hereinafter referred to as aminated oligo DNA) was synthesized in which an amino group was introduced at the 5'end of oligo DNA having the sequence ATTTGCGGTGGACGATG-3 '.

(Preparation of Rhodamine-Labeled Oligo DNA) 5'-CA paired with the base sequence of the aminated DNA described above.
TCGTCCACCCGCAAATGCTTCTA-3 '
The oligo DNA having the 5'end of the oligo DNA having the sequence
(Referred to as A) was synthesized.

(Fixed) aminated oligo DNA was added to Ald
Eyde Spotting Solution (G
Dissolved in ENPAK) at a concentration of 0.5 mg / ml,
A DNA spot solution was prepared. Use a DNA chip spotter (manufactured by Nichiryo Corporation) to display DNA on each substrate.
Spot solution, spot at 37 ℃ for 30 minutes, 80 ℃ for 60 minutes
Heat for minutes and use ethanol 1 as a blocking solution.
0.5 g NaB in 3.3 ml and PBS (-) 45 ml
H4 was dissolved and prepared, and the substrate was immersed in this blocking solution for 5 minutes, washed with pure water, further treated in boiling water for 3 minutes, immersed in ice-cooled ethanol for 1 minute, and air-dried.

0.2% of rhodamine-labeled oligo DNA
A rhodamine-labeled oligo DNA solution dissolved in 5 × SSC solution containing SDS was prepared, boiled for 3 minutes, cooled with ice, and 80 μl of this solution was dropped onto a substrate on which aminated oligo DNA was immobilized, and the solution was covered with a cover glass. Cover, moisturize 60
After incubating at 18 ° C. for 18 hours, the cover glass was removed, washed with 2 × SSC containing 0.5% SDS, 0.5 × SSC, and pure water in this order, air-dried, and provided for comparison of the amount of immobilized DNA.

The amount of DNA immobilized was compared by taking a fluorescence image of rhodamine with a fluorescence microscope (manufactured by Olympus Co., Ltd.), focusing on each spot, setting a common exposure time, and taking a photograph of the fluorescence image. The image was developed under various conditions, the photograph was read as image data by an image scanner, the fluorescence intensity was quantified by image processing on a computer, and the amount of immobilized aminated oligo DNA was compared. The average value of each spot in Example 1 was set to 100, and the immobilization amount of each substrate was compared.

(3) Reliability: A slide glass molded product (L76 mm × W26 mm × t1 mm) has a height of 50 c.
The highest heights at which the substrate did not break by free fall from m, 100 cm, 150 cm, and 200 cm are shown in the table.

[0034]

[Table 1]

[0035]

INDUSTRIAL APPLICABILITY As described above, the substrate of the present invention has excellent flatness, and it is possible to immobilize and detect target DNA on the substrate without any special treatment, and DNA is more reliable than
It is useful for chip substrates.

Claims (3)

[Claims] 1. A microchip substrate comprising at least an epoxy resin (a) represented by formula 1, an epoxy resin (b) represented by formula 2, and a curing agent. [Chemical 1] (M = 0 or more integer) (N is an integer of 0 or more, R is a hydrocarbon residue having 1 to 10 carbon atoms (may include a functional group such as alcoholic OH)). 2. The microchip substrate according to claim 1, wherein the content of the epoxy resin (b) is 2 to 20 gram equivalents relative to 100 gram equivalents of the epoxy resin (a). 3. A microchip using the substrate for microchip according to claim 1 or 2.