WO2003046562A1 - Plastic substrate for microchips - Google Patents

Abstract

A plastic substrate for microchips characterized in that an aminoalkylsilane having a glutaraldehyde-origin aldehyde group introduced into an amino group exists on the surface, a process for producing the same and a method of using the same. In case of fixing DNA fragment chains, the above substrate shows little scattering in the amounts of the fixed DNA fragment chains, enables highly efficient and highly reproducible hybridization and has excellent storage properties.

Description

 Light

Technical field

 The present invention relates to a plastic substrate for a microchip having high fixing efficiency of DNA fragment chains and high efficiency of pre-dips, a method for producing the same, and a method for using the same. Rice field

Background art

 The method of immobilizing a single-stranded DNA fragment on a substrate is roughly classified into an adsorption method and a method of forming a covalent bond.

 The DNA is immobilized on the substrate by adsorption of the DNA fragment chain, utilizing the fact that DNA has a negative charge.A positive charge is applied to the substrate surface, and the DNA fragment is adsorbed and immobilized by electrical attraction. Is what you do.

 As a substrate used in such a method, a substrate coated with polylysine on its surface is used. However, polylysine generally has poor stability after coating and is difficult to store for long periods. The ability to immobilize DNA fragments decreases over time after polylysine is coated on a substrate. Furthermore, the DNA has poor storage stability after immobilization, and can only be stored for about two weeks at room temperature.

 Similarly, as a method for imparting a positive charge, there is a method of reacting a silane coupling agent having an amino group with a glass substrate to introduce an amino group.

 Conventional substrates use glass, and silane coupling agents that are compatible with glass are used. The reaction of the silane coupling agent on the glass substrate is easy. If a silane coupling agent having an amino group is used, the amino group can be easily introduced into the surface of the glass substrate. ,

When immobilizing long-chain DNA consisting of hundreds to thousands of bases, amino groups are introduced. It is effective to perform adsorption using the fact that the DNA chain has a negative charge.Introduction of an amino group using an aminosilane coupling agent is an effective method with high storage stability as an alternative to the above-mentioned polylysine coat. It is a way.

 However, it is difficult to apply the method to immobilization of a short DNA chain called oligo DNA, which is composed of several tens of bases.

 This is because, in the case of short DNA strands, stable adsorption of DNA cannot be achieved by simple adsorption alone, resulting in dropout of DNA after spotting on a substrate and detection of DNA by hybridization. This is because hybridization does not occur efficiently and DNA cannot be detected, or the detection intensity varies.

 Therefore, a method of forming a covalent bond and fixing the DNA to the substrate surface has been adopted. The most well-known method is to use an immobilization substrate on which an amino group has been introduced with a silane coupling agent as described above, while introducing an amino group at the end of the DNA fragment chain, and using a method such as daltaraldehyde. In this method, a DNA fragment chain is covalently immobilized on the substrate surface using a crosslinking agent.

 In the case of a glass substrate, the surface has many hydroxyl groups, and the introduction density of a silane coupling agent having an amino group becomes high. The introduction of a high density of amino groups also appears to be effective in immobilizing DNA fragment chains with many attachment points. However, as described above, in the formation of a covalent bond by cross-linking with daltaraldehyde, when the density of amino groups derived from the silane coupling agent is high, the addition of dartartaldehyde can result in the formation of the silane coupling agent. Amino groups are cross-linked, so that covalent bonds cannot be formed with amino groups on the DNA strand side, and the amount of immobilized DNA fragment chains also decreases.

It is also conceivable to dilute the concentration of the silane coupling agent to lower the density of amino groups moderately.However, if there is a free hydroxyl group without reacting with the silane coupling agent, the charge on the entire surface will be increased. Is negatively charged and has a negative charge The DNA chain becomes difficult to approach the surface, hinders the bond with the aldehyde group introduced at the tip of the amino group, and it becomes difficult to fix a sufficient amount of the DNA fragment chain.

 For the above reasons, it has been difficult to obtain a substrate suitable for a Stanford-style DNA chip with high efficiency in DNA immobilization and hybridization on a glass substrate and high reproducibility.

 An object of the present invention is to use as a substrate for use in the production of a DNA chip, so that the efficiency of immobilizing DNA fragment chains is high, and the amount of immobilized DNA fragment chains on the entire substrate has no variation and is further immobilized. To provide a substrate for immobilizing DNA with high hybridization efficiency and high reproducibility in the hybridization between the DNA fragment strand and the target DNA strand, and stably maintain the DNA even after the substrate is manufactured. It is an object of the present invention to provide a high quality preserving substrate for fixing a DNA, which can fix a fragment chain and perform eight hybridizations. Disclosure of the invention

 In order to solve the conventional problems as described above, the present invention has conducted intensive studies. As a result, the surface of a plastic substrate was subjected to a hydrophilic treatment to introduce an amino group with an aminoalkylsilane, and then the daltal was added to the amino group. When an aldehyde group was introduced with an aldehyde, it was found that the fixation efficiency of the DNA fragment was high and reproducible, that the quality of the plastic substrate was stable even after the introduction of the aldehyde group, and that the reactivity of the aldehyde group was maintained. The present invention has been completed.

 That is, the present invention provides a plastic substrate for a microchip, characterized in that an aminoalkylsilane having an aldehyde group derived from dartalaldehyde introduced into the amino group is present on the surface.

 Also, the present invention relates to a plastic substrate

(1) oxidation treatment of the surface, (2) contact with a solution containing aminoalkylsilane, and

 (3) Contact with a solution containing daltaldehyde

The present invention provides a method for producing a plastic substrate for microchips, wherein an aminoalkylsilane in which an aldehyde group derived from dartartaldehyde is introduced into an amino group is present on a substrate surface by a step including:

 Further, in the present invention, a DNA chain having an amino group introduced into a terminal is dissolved in a solution, and this solution is brought into contact with the above-mentioned plastic substrate for microchips to introduce the amino group of the DNA chain and the substrate. It is intended to provide a method of using a plastic substrate for microchips, wherein a DNA chain is immobilized on a substrate by covalently bonding to an aldehyde group. Brief description of the drawings

 FIG. 1 is a conceptual diagram showing one embodiment of the substrate shape of the present invention.

 FIG. 2 is a conceptual diagram showing another embodiment of the substrate shape of the present invention.

 FIG. 3 is a conceptual diagram showing another embodiment of the substrate shape of the present invention.

 FIG. 4 is a conceptual diagram showing another embodiment of the substrate shape of the present invention.

 FIG. 5 is a histogram showing the distribution of the amount of background fluorescence in Reference Example 4 and Comparative Reference Example 5.

 In FIGS. 1-4, 1 is the back surface of the sample fixing surface, and 2 is the thick portion. BEST MODE FOR CARRYING OUT THE INVENTION

The plastic substrate for a microchip of the present invention is characterized in that aminoalkylsilane having an amino group to which an aldehyde group derived from aldehyde is introduced is present on the surface. Examples of the aminoalkylsilane include aminoalkyltrialco. For example, the following formula (1)

(In the formula, n represents an integer of 1 to 16, and RR ² and R ³ each represent an alkyl group.)

The compound represented by is preferred. In the formula (1), n is preferably from 1 to: L 0, particularly preferably from 1 to 8. The alkyl group represented by R ² and R ³ is preferably an alkyl group having 1 to 6 carbon atoms, particularly an alkyl group having 1 to 4 carbon atoms such as a methyl group, an ethyl group, and a propyl group.

Daltaraldehyde (0HC (CH ₂ ) ₃ CH0) has been introduced into the amino group of the aminoaminosilane. That is, the aldehyde and the amino group form a Schiff base (-CH = N_) or a reduced form thereof (-CM_).

 Moreover, on the substrate surface of the present invention, the aldehyde group-introduced aminoalkylsilane may be mixed with an aldehyde group-introduced aminoalkylsilane. Here, as the aminoalkylsilane into which the aldehyde group has not been introduced, those represented by the above general formula (1) can be mentioned.

The substrate surface of the present invention may further contain alkylsilane. Here, as the alkylsilane, for example, the following formula (2)

(In the formula, m represents an integer of 1 to 16, and R ⁴ , R ⁵ and R ⁶ each represent an alkyl group.)

The compound represented by is preferred. In the formula (2), m is preferably 1 to 8, particularly preferably 1 to 6. The alkyl group represented by R ⁴ , R ⁵ and R ⁶ is preferably an alkyl group having 1 to 6 carbon atoms, particularly an alkyl group having 1 to 4 carbon atoms such as a methyl group, an ethyl group and a propyl group.

Looking at the surface of the substrate of the present invention at the molecular level, the molecular chain having an aldehyde group at the tip is It is considered that the molecular chains (reactive aminosilanes and / or alkylsilanes) having no reactive groups are in a form standing from the substrate surface. When aminoalkylsilane and alkylsilane are mixed, the mixing ratio is adjusted to maintain the distance between amino groups sufficiently and prevent the cross-linking of amino groups by glutaraldehyde to efficiently introduce aldehyde groups. Is done. '

 When aminoalkylsilane and alkylsilane are mixed, the mixing ratio depends on the amount of hydroxyl groups introduced into the substrate.Alkylsilane is 100 to 100 parts by weight, and alkylsilane is 20 to 400 parts by weight. The range of parts is preferred. When the amount is less than 20 parts by weight, the effect of mixing the alkylsilane is not recognized. When the amount is more than 400 parts by weight, the density of the aldehyde group becomes low, and the amount of immobilized DNA per unit area decreases.

 The mixing ratio of alkylsilane is adjusted according to the length of DNA to be finally hybridized. If the DNA length is long, it is more effective to increase the ratio of the alkylsilane, and if it is short, it is more effective to increase the mixing efficiency of the alkylsilane to increase the high-purity efficiency.

The molecular chain of the aminoalkylsilane and the alkylsilane is preferably linear, and those having a bent nitrogen atom or the like in the middle thereof are not preferred. If a bent molecular chain is used, the above-described state in which the molecular chains are supported by each other cannot be formed, and the DNA immobilization efficiency is greatly reduced. Furthermore, by using an alkylsilane having a shorter molecular chain length than the aminoalkylsilane, the molecular chain having the aldehyde group comes out of the head while the molecular chain having the aldehyde group is supported by the molecular chain of the alkylsilane. Because of this, the DNA fragment having the aldehyde group and the amino group introduced therein efficiently reacts, and a high DNA immobilization rate can be obtained. Further, the immobilized DNA strand is immobilized while keeping a distance from the substrate surface, so that the DNA fragments are likely to be homologous in the subsequent hybridization step, thereby increasing the hybridization efficiency. The substrate of the present invention is a plastic substrate. The plastic used for this substrate is not particularly limited as long as it has good moldability, water resistance, and heat resistance.However, there is a method for detecting DNA by modifying fluorescence with DNA. A material that is widely used and preferably generates less fluorescence is preferable. Examples of such a resin include a saturated cyclic polyolefin resin and a fluororesin.

 Among them, saturated cyclic polyolefins are preferable because of their high heat resistance and little autofluorescence in the wavelength region of Cy3 and Cy5, which are fluorescent dyes widely used in DNA chips. Fluororesins have low autofluorescent power S over a wide wavelength range, but have the disadvantage that they require removal of fluorine generated during molding and are difficult to mold. As the saturated cyclic polyolefin-based resin, a saturated polymer obtained by hydrogenating a polymer having a cyclic olefin structure or a copolymer of a cyclic olefin and a Hi-Iseki olefin is preferable.

Examples of the former include, for example, hydrogenated products of a ring-opened polymer of norpolene represented by the general formula (3).

(However, in the above formula (3), R ⁷ and R ⁸ may be hydrogen or a hydrocarbon residue having 1 to 10 carbon atoms, which may be the same or different, and R ⁷ and R ⁸ are each a ring. May be formed.)

The polymer having the structural unit represented by the general formula (3) is norpolene and its alkyl or alkylidene-substituted monomer, and specifically, 5-methyl-12-norporene, 5,6-dimethyl-2 —Norbornene, 5-ethylidene-2-norbornene, etc. Other than these, it is obtained by ring-opening polymerization using dicyclopentene, 2,3-dihydroxycyclopentene and their alkyl-substituted products such as methyl and ethyl. There is a saturated polymer produced by hydrogenating a ring-opening polymer. You.

 Further, a polymer of a cyclic olefin monomer represented by the general formula (4), or an α-amine such as ethylene, propylene, isopropylene, 1-butene, 3-methyl-1-butene, 11-pentene, or 11-hexene. A saturated polymer produced by hydrogenating a random copolymer of olefin and a cyclic olefin monomer represented by the general formula (4) may be used.

(However, in the above formula (4), R ^{9 to} R ¹⁶ are each selected from the group consisting of hydrogen, a halogen atom and a hydrocarbon residue, and R ^{13 to} R ¹⁶ may form a ring with each other.) Further, a polymer of a cyclic olefin monomer represented by the general formula (5), or ethylene, propylene, isopropylene, 1-butene, 3-methyl-1-butene, 11-pentene, 1-hexene A saturated polymer produced by hydrogenating a random copolymer of α-olefin and a cyclic olefin monomer represented by the general formula (5) may be used.

(However, in the above formula (5), R ^{9 to} R ^2D represent hydrogen, halogen atom and hydrocarbon, respectively) Selected from the group consisting of Motozanmoto, R ^{I 7} ~R ² are each may form a ring. p represents a number of 1 or more. )

 Among them, a saturated cyclic polyolefin resin containing at least one of norpolene or a norpolene derivative (for example, a compound of the formula (3)) as a monomer unit is particularly preferable.

 The form of the substrate of the present invention is not particularly limited as long as the form is generally used for immobilizing a biological substance and used for measurement. For example, plates for immunoassay widely used in the field of immunoassay and beads similarly can be mentioned, and a plate-like form used as a DNA chip is most preferable.

 The substrate of the present invention is also used in a method of detecting a sample on which a process to which a fluorescent label has been added has been performed by using fluorescence emitted by supplying excitation light. In such a case, the plastic used for the substrate contains pigment to prevent the invasion of the excitation light received by the substrate and to prevent the fluorescence emitted by the excitation light from reaching the detector. Preferably, it is opaque.

 The pigment may be either a black pigment or a white pigment, but preferably includes a black pigment, and more preferably includes carbon black in terms of colorability, heat resistance, bleed resistance, and chemical resistance. .

In order to detect a fluorescent label attached to a very small amount of sample on a microchip, a method of irradiating a strong laser beam as excitation light to increase the amount of fluorescence is often used. Less than 1% by weight of the pigment incorporated in the plastic is not sufficiently opaque, and the laser light does not block and penetrates into the plastic, and the background fluorescent noise generated by the plastic leaks out. However, it is detected as a sufficiently large value for the signal of the fluorescent label. For this reason, at least 1% by weight of pigment is required to prevent laser light from penetrating from the surface of the substrate into the interior and to prevent fluorescence generated in the surface molecular layer of the substrate from leaking from the surface molecular layer. Increasing the pigment content reduces the number of surface molecular layers on the substrate that leaks fluorescence, It is also possible to reduce the amount of fluorescent light.

 If the pigment is added in an amount of more than 60% by weight, the amount of plastic contained in the substrate will be reduced, and the plastic's inherent moldability, hardness, heat resistance, wettability, sample adsorption, and other physical properties It is unsuitable because of the significant change in its chemical properties, which may cause problems in use. As long as the resin is blended in at least 60% by weight or less, it is expected that the physicochemical properties of the plastic will not change much. Naturally, the smaller the pigment content, the smaller the change in physicochemical properties compared to the original plastic, which is preferable in designing a microchip.

 In consideration of the above points, the content of the pigment contained in the plastic of the substrate is 1 to 60% by weight, preferably 1.5 to 20% by weight, and more preferably 2 to 5% by weight. The physicochemical properties of plastics are not practically changed, and the fluorescence generated from the plastics is reduced and the signal of the fluorescent label can be detected clearly.

 The substrate of the present invention is provided with means for limiting the contact surface with the measurement device on the back surface of the substrate in order to prevent deformation of the sample fixing surface due to uneven force applied from the substrate fixing jig of the measurement device. Is preferred. As the means for limiting the contact surface, it is preferable to provide a convex portion on a part of the back surface of the sample fixing surface, and it is more preferable that the convex portion be an outer edge portion that is thicker than the thickness of the sample fixing surface.

 The sample fixing surface of the substrate of the present invention will be described in more detail. When using a scanner with a confocal detection system for evaluation, it is difficult to focus if deformation such as distortion or warpage occurs on the sample fixing surface of the DNA chip substrate, and accurate data can be obtained. Absent. Many of the causes of substrate deformation are the force received from the substrate fixing jig in the scanner. In order to avoid deformation due to the force from the jig, (A) increase the rigidity of the substrate, (B) make the structure in which the sample fixing surface does not directly contact the jig, and transfer the force to the sample fixing surface. Minimizing, etc.

Specific methods of (A) include increasing the thickness of the substrate and kneading the filler. To increase the strength of the material itself. However, increasing the thickness of the substrate may have serious adverse effects such that conventional equipment cannot be used. In addition, the method of kneading the filler has problems such as accelerated abrasion of the molding die and change in surface treatment properties, which is not practically preferable. On the other hand, the above-mentioned problem does not occur in the method (B). That is, since the thickness of the entire substrate is the same as that of the conventional product, there is no need to switch between devices and equipment, and there is no problem in terms of manufacturing because the substrate material itself is unchanged.

 The shape of the jig for fixing the substrate of the scanner varies depending on the model, but the most commonly used method is to push up the lower surface of the substrate with a panel panel and fix it by pressing it on the upper jig. In this method, if the pushing force of the panel is too high, the central part of the substrate may be pushed up, causing distortion on the measurement surface. Therefore, an area that protrudes beyond the sample fixing surface is provided at the outer edge of the substrate, and contact with the jig is limited to only the protruding portion, thereby eliminating uneven stress application to the sample fixing surface and avoiding deformation. can do. As for the shape of the protruding portion, as shown in FIGS. 1 to 4, it is preferable to provide a frame-shaped region at the outer edge of the back surface of the sample fixing portion, and to make this portion thicker than the sample fixing surface. By increasing the thickness of the outer edge, the rigidity of this area increases, and the majority of the force from the substrate fixing jig is borne.Therefore, the stress applied to the sample fixing surface decreases, and deformation can be avoided. Become.

 Since the deformation due to the force from the substrate fixing jig is about 20 to 100 m, the thickness difference between the sample fixing portion and the outer edge is preferably 20 to 500 m, more preferably 20 to 200 zm, and most preferably 20 to: L00m.

Further, by adopting the above shape, an effect of preventing the sample fixing surface from being damaged when handling the substrate can be obtained. In other words, in the case of a normal flat substrate, when the substrate is placed on a workbench or the like, small scratches may be made on the sample fixing surface or the back surface, and this may be detected as noise during measurement. Since the substrate of the present invention does not come into direct contact with the substrate, the possibility of scratching is reduced, and highly accurate measurement can be performed. Hereinafter, the plastic substrate for microchip of the present invention, a method for producing the same, and a method for using the same will be described in detail.

 The substrate of the present invention is a plastic substrate,

 (1) oxidation treatment of the surface,

 (2) contact with a solution containing aminoalkylsilane, and

 (3) Contact with a solution containing daltaldehyde

The process is carried out by causing an aminoaminosilane in which an aldehyde group derived from dartartaldehyde is introduced into the amino group to be present on the substrate surface. The oxidation treatment of the surface of the substrate is a step of introducing a hydroxyl group into the surface of the substrate. Low-temperature plasma treatment, corona discharge treatment, flame treatment, and other chemical treatments can be cited as a method of the acidification treatment. Low-temperature plasma treatment can be performed to stably and uniformly oxidize the resin surface. Is preferably used, and oxygen is preferably used as a gas species to be used in this case because a stable introduction of a hydroxyl group can be performed. Therefore, it is preferable to carry out the treatment in the oxygen or gas atmosphere containing oxygen in the acid treatment.

Immediately after the introduction of the hydroxyl group on the surface of the substrate by the above-mentioned method, it is preferable to further introduce the hydroxyl group into a radical state or a T-bonded carbon atom. The method of introducing hydroxyl groups at this stage is preferably one that has a chance to come into contact with water molecules during treatment and post-treatment, and is immersed in a solution such as a dilute aqueous solution of permanganate, a mixed solvent of alcohol and water, or pure water. Immersion in pure sulfuric acid, followed by immersion in pure water, or contact with an atmosphere of 80 to 100% humidity. Among them, a method of immersing in pure water which is simple and is not limited in shape and has no problem of disposal is most preferable. When oxygen gas low-temperature plasma discharge treatment is performed as the oxidation treatment, it is expected that carbon atoms on the substrate surface will form a radical state of carbon atoms and 7T bonds due to oxygen radicals. Other types of resins with high light transmittance, such as polystyrene and polycarbonate, originally contain an aromatic ring in the molecular structure of the polymer, so the fluorescence of the resin itself is strong, and it does not pose a problem as fluorescence noise that increases due to oxidation. Was. However, the molecular structure of a saturated cyclic polyolefin resin such as a norpolene resin does not include the 7T binding force, and the fluorescence of the original resin itself is very small. When oxygen gas low-temperature plasma discharge treatment is performed, about 10 to 25% of carbon atoms present on the substrate surface have 7T bonds, which is a factor that increases the fluorescence of the substrate itself due to oxygen gas low-temperature plasma discharge treatment. It is thought that it became.

 The increase in the fluorescence of the substrate itself due to the oxygen gas low-temperature plasma discharge treatment is due to the introduction of hydroxyl groups into radical carbon atoms by immersion in pure water immediately after the oxygen gas low-temperature plasma discharge treatment. It is possible to suppress the increase. If the ratio of carbon atoms having a 7C bond in the molecular layer constituting the surface of the substrate is 15% or less, noise does not hinder measurement due to autofluorescence. Accordingly, the ratio of carbon atoms having a 7t bond in the molecular layer constituting the surface of the substrate is preferably 15% or less, and more preferably 10% or less.

 In addition, by performing the above-described treatment, a large amount of hydroxyl groups can be introduced into the substrate surface, and the effect of increasing the number of reactive sites in applying the surface treatment to the substrate can be obtained. For example, in the case of coating with an aminosilane agent as disclosed in Japanese Patent Application Laid-Open No. 60-155600, the more amino groups are present on the substrate surface, the more the aminosilane agent is coated. It is possible. The substrate immersed in pure water immediately after the oxygen gas low-temperature plasma discharge treatment is present in the molecular layer that forms the surface of the substrate, compared to the case where the oxygen-gas low-temperature plasma discharge treatment is performed only on the substrate of the saturated cyclic polyolefin resin. About 1.5 times more hydroxyl groups can be introduced.

Next, the amino group is brought into contact with the aminoalkylsilane to react the hydroxyl group introduced on the surface of the substrate with the aminoalkylsilane. A solution in which an aminoalkylsilane is dissolved in an organic solvent such as methanol is prepared, and a substrate having a surface treated with acid is immersed in the solution and allowed to stand, thereby introducing an amino group to the substrate surface. After the reaction, the substrate is taken out of the solution and washed. Here, instead of aminoalkylsilane Lusilane and alkylsilane are introduced into the substrate surface in a mixed form.

 Next, an aldehyde group is introduced into the amino group. Daltaraldehyde is dissolved in the solution, and the carrier having the amino group introduced therein is immersed in the solution and allowed to stand. One aldehyde group of the darthal aldehyde is reacted with the amino group, and after standing, washed with ultrapure water. After drying, a substrate having an aldehyde group introduced is finally obtained. Here, in the case where amino silane and alkyl silane are mixed in the substrate, the plastic substrate in which aminoalkyl silane and alkyl silane in which the aldehyde group derived from dartaldehyde is introduced into the amino group is mixed on the surface. Get.

 Next, a method for immobilizing DNA using the present invention will be described.

 As the DNA to be immobilized on the substrate surface, an oligo DNA consisting of several tens of base chains is appropriate. In that case, an amino group is introduced at the end of the DNA chain.

 The amino group-introduced DNA chain is dissolved in a DNA fixing solution, spotted on the substrate by a machine called a spotter, and fixed by standing.

 Conventionally, glutaraldehyde must be added to a solution on a substrate into which an amino group has been introduced when DNA chains are immobilized.However, in the substrate of the present invention, daltalaldehyde is introduced. There is no need to add daltaldehyde in the solution.

 If dartartaldehyde is added to the DNA solution, the amino groups introduced at the ends of the DNA chains will cross-link, reducing the number of DNA chains that can be actually fixed. Since dartartaldehyde is not added to the DNA, the size of the DNA fragment chain to be immobilized can be suppressed.

In the method of the present invention, when an aldehyde group is introduced, when a saturated cyclic polyolefin resin is used as a resin constituting the substrate, the amount of the aldehyde group introduced is large, and several tens to about 50, which is called oligo DNA, is used. The immobilization rate of DNA strands consisting of bases is high, and the detection efficiency of DNA to be detected in hybridization is also high. The detection efficiency of DNA hybridization is that the DNA to be detected is a long DNA strand with a base number of about 50,000 to 100,000. An appropriate distance is required. In the case of a plastic surface, particularly a surface of a saturated cyclic polyolefin resin, the distance is considered to be the most suitable distance for performing hybridization.

 In addition, it was found that even if the aldehyde was introduced and stored for a long time, the DNA immobilization ability did not decrease. This is considered to be because the density of the introduced amino groups is in an appropriate state, and crosslinking between the amino groups is unlikely to occur. Example

 Hereinafter, the present invention will be specifically described with reference to examples.

 (Example 1)

 A slide glass substrate was obtained by injection molding using a saturated cyclic polyolefin resin (a hydrogenated product of a random copolymer of ethylene and dicyclopentadiene, a norbornene derivative). The surface of this molded product was subjected to hydrophilic treatment by low-temperature oxygen plasma treatment. Next, aminoaminosilane was prepared by dissolving aminopropyltriethoxysilane at a concentration of 5% in methanol to prepare an amino group-introducing treatment solution. After dipping in this solution for 2 hours, the substrate was removed. The substrate was taken out of the solution, immersed in ultrapure water, allowed to stand, and the substrate was taken out and dried. Daltaraldehyde PBS

(1) Daltaraldehyde solution was prepared by dissolving it in a concentration of 2%, and the substrate subjected to the aminoalkylsilane treatment was immersed in the daltaraldehyde solution, allowed to stand for 4 hours, and then taken out of the substrate and taken out. It was immersed in pure water, washed and dried.

 (Example 2)

After the substrate manufactured in Example 1 was put in a case for a slide glass, it was put in aluminum, PET, and a laminate bag, the bag was sealed, and stored at room temperature for 6 months. (Comparative Example 1) A glass slide glass was prepared, and a solution prepared by dissolving it at a concentration of 5% in methanol using aminopropyltriethoxysilane as an aminosilane coupling agent was prepared as an amino group-introducing treatment solution. After immersion for 2 hours, the substrate was taken out of the solution, immersed in ultrapure water, allowed to stand, and then taken out and dried. Daltaraldehyde is dissolved in PBS (-) at a concentration of 2% to prepare a daltaraldehyde solution.The aminosilane-treated substrate is immersed in the daltaraldehyde solution, and left for 4 hours. Removed, immersed in ultrapure water, washed and dried

 (Comparative Example 2)

 After placing the substrate manufactured in Comparative Example 1 in a case for a slide glass, the substrate was placed in aluminum, PET, and a laminate bag, the bag was sealed, and stored at room temperature for 6 months. (Aminated oligo DNA)

An oligo DNA having an amino group introduced at the 5 'end of the oligo DNA having the sequence of 1) (hereinafter referred to as aminated oligo DNA) was synthesized.

 (Mouth Damin Label Oligo D NA)

 The oligo DNA having the sequence of 5'-CATCGTCCACCGCAAATGCTTCTA-3 '(SEQ ID NO: 2), which is labeled with rhodamine at the 5' end of the oligo DNA which is paired with the nucleotide sequence of the aminated DNA (hereinafter referred to as rhodamine-labeled oligo) DNA).

 (Cy 3 labeled cDNA)

 sense

3) One 3 '(SEQ ID NO: A primer consisting of the sequence of 1) was synthesized, while cDNA was procured from HeLa cells. Using this cDNA and the primers described above, a cDNA corresponding to 661 actin / 3/3 actin (hereinafter referred to as Cy3-labeled cDNA) was synthesized by PCR using Cy3.

 (Comparison of DNA immobilization efficiency)

The aminated oligo DNA was dissolved in Aldehyde Spotting Solution (GENPAK) at a concentration of 0.5 mg / mL to prepare a DNA spot solution. Spot the DNA spot solution on each substrate using a DNA chip spotter (Nichiriyo Co., Ltd.), heat at 37 ° C for 30 minutes and at 80 ° C for 60 minutes, and add 13.3 mL of ethanol as a blocking solution. PBS (-) and N a BH ₄ of 0. 5 g prepared by溶角early in 45 mL, the substrate was washed with pure water After immersed for 5 minutes in the blocking solution, and et 3 minutes treatment in boiling water After that, it was immersed in ice-cooled ethanol for 1 minute and air-dried.

 A rhodamine-labeled oligo DNA solution prepared by dissolving rhodamine-labeled oligo DNA in a 533 solution containing 0.2% 303 is prepared, boiled for 3 minutes, and cooled on ice. 80 xl is dropped on the substrate on which is fixed, covered with a glass cover, incubated at 60 ° C for 18 hours in a moisturized condition, and a cover glass is taken out.2 XSSC, 0.5 XSSC containing 0.5% SDS, pure water After washing in the order described above, the sample was air-dried and used for comparison of the amount of DNA immobilized.

Comparison of the amount of DNA immobilization was performed by using a fluorescence microscope (Olympus), taking a fluorescence image of rhodamine, focusing on each spot, and taking a picture of the fluorescence image with the same exposure time, etc. Developed under the conditions, the photograph was read as image data by an image scanner, the fluorescence intensity was digitized by image processing on a computer, and compared as the immobilized amount of aminated oligo DNA. The average value of each spot in Example 1 was set to 100, and a comparison of the amount of immobilization of each substrate and a comparison of the intensity variation between spots were made. Table 1 shows the results. (Comparison of detection intensity in hybridization)

 The aminated oligo DNA was dissolved in Aldehyde Spotting Solution (GENPAK) at a concentration of 0.5 mg / iiiL to prepare a DNA spot solution. Spot the DNA spot solution on each substrate using a spotter for DNA chips (produced by Nichiriyo Co., Ltd.), heat at 37 ° C for 30 minutes and at 80 ° C for 60 minutes, and use 13.3 mL of ethanol as a blocking solution. Dissolve 0.5 g of Na BH4 in 45 mL of PBS and PBS (-), prepare the substrate, immerse the substrate in this blocking solution for 5 minutes, wash with pure water, and further treat in boiling water for 3 minutes After immersion, it was immersed in ice-cooled ethanol for 1 minute and air-dried.

 Prepare a Cy3-labeled cDNA solution in which Cy3-labeled cDNA is dissolved in 5XSSC solution containing 0.2% SDS, boil for 3 minutes, cool on ice, and place this solution on the substrate on which the aminated oligo DNA is immobilized. , Cover with a cover glass, incubate at 60 ° C for 18 hours under moisturization, remove the cover glass, wash in 2XS SC containing 0.5% SDS, 0.5 XSSC, pure water, and air-dry. For comparison of the amount of DNA immobilized.

 The comparison of the amount of DNA immobilization was performed by using a fluorescence microscope (Olympus), taking a fluorescence image of rhodamine, focusing on each spot, taking a picture of the fluorescence image with the same exposure time, etc. After developing under the conditions, the photographs were read as image data using an image scanner, and the fluorescence intensity was quantified by image processing on a computer, and compared as the amount of immobilized hybridized cDNA. The average value of each spot in Example 1 was set to 100, and the immobilization amount of each substrate was compared and the intensity variation between spots was compared. Table 2 shows the results.

 Table 1 Comparison of amount of oligo DNA immobilized

 Average value Maximum value Minimum value CV value (%)

 Example 1 100 121 86 13.5

 Example 2 95 112 85 14.5

 Comparative Example 1 55 70 30 19.5

Comparative Example 2 32 40 15 25.3 Table 2 Comparison of the amount of hybridized cDNA

表 1及び表 2から明らかなように、 本発明の基板は固定化効率が高く、 力、つ長 期の保存においても固定化能の低下が認められず、 また、 D NAのスポッティン グによる D NAの固定化において、 スポット内の D NAの固定が均一であり、 ハ ィブリダイズ後の D N Aの検出におけるスポットの検出強度のバラツキが少な い。

As is evident from Tables 1 and 2, the substrate of the present invention has a high immobilization efficiency, does not show a decrease in the immobilization ability even when stored for a long period of time, and has a high D value due to spotting of DNA. In the immobilization of NA, the immobilization of DNA in the spot is uniform, and the variation in detection intensity of the spot in the detection of DNA after hybridization is small.

 ■ Furthermore, in fixing DNA, it is not necessary to add daltaraldehyde to the solution, so that the loss of DNA due to cross-linking of DNA chain fragments can be suppressed to a low level.

 (Example 3)

 Using the same saturated cyclic polyolefin resin as in Example 1, a slide glass-shaped substrate was obtained by injection molding. The surface of this molded product was subjected to a hydrophilic treatment by a low-temperature oxygen plasma treatment. Next, a solution prepared by dissolving aminopropyltriethoxysilane and methyltriethoxysilane at a concentration of 5% in methanol at a ratio of 2: 1 was prepared as an amino group introduction treatment solution, and immersed in this solution for 2 hours. After crushing, the substrate was taken out of the solution, immersed in ultrapure water, allowed to stand, and then taken out and dried. Daltaraldehyde is dissolved in PBS (1) at a concentration of 2% to prepare a daltaraldehyde solution, and the substrate treated with aminoalkylsilane is immersed in the daltaraldehyde solution and left for 4 hours. Then, the substrate was taken out, immersed in ultrapure water, washed and dried.

Using the substrates obtained in Examples 1 and 3 and Comparative Example 1, a comparison of the amount of immobilized DNA and a comparison of the detection intensity in eight hybridizations were performed in the same manner as described above. The results are shown in Tables 3 and 4. The values in Tables 3 and 4 are based on the data of Example 3. It is shown as 100.

表 4 c D NAのハイブリダィズ量の比較

表 3及び 4より、 ァミノアルキルシランに加えてアルキルシランをプラスチッ ク基板表面に存在せしめると、 D N A固定化効率及びハイブリダイズ効率がさら に向上することがわかる。 Table 3 Comparison of the amount of immobilized oligo DNA

Table 4 Comparison of hybridization amount of cDNA

Tables 3 and 4 show that the presence of alkylsilane on the surface of the plastic substrate in addition to aminoalkylsilane further improves the DNA immobilization efficiency and the hybridization efficiency.

 (Reference Example 1) A slide was injection-molded using a hydrogenated substance to obtain a molded product A. This was oxidized for 10 minutes by generating high-frequency low-temperature plasma while passing oxygen gas under reduced pressure. Immediately thereafter, immersion treatment was performed for 10 seconds in pure water.

 (Comparative Reference Example 1)

 The molded article A of the reference example was subjected to an acid treatment for 10 minutes by generating high-frequency low-temperature plasma while passing oxygen gas under reduced pressure.

 [1] Autofluorescence

 The slides of Reference Example 1 and Comparative Reference Example 1 were observed with an epi-illumination type fluorescence microscope (excitation light wavelength: 532 nm, fluorescence wavelength: 560 nm). Fluorescence was photographed with a CCD camera, and the amount of fluorescence was compared and evaluated based on the image data.

Table 5 shows the evaluation results. From this, the saturated cyclic polyolefin-based resin forms It was found that the background fluorescence could be suppressed by introducing a hydroxyl group into the carbon atom.

 [2] ESCA analysis

ESCA (model: ESCALAB 220i-XL, manufactured by F1 SURFACE SYSTEMS, analysis area: 0.6 mm, photoelectron escape angle: 90 deg, degree of vacuum: 1. 5 X 10- ⁶ Pa, X-ray source: a l Ko; line) was surface analyzed by, were compared and evaluated the percentage another bonding state of carbon atoms that exist in the molecular layer of the surface of the substrate. Table 6 shows the evaluation results. Thus, it was recognized that the application of the formulation of the present invention increased the number of hydroxyl groups and decreased the number of carbon atoms having a π bond.

表 6 Table 5

Table 6

参考例 1の基板を用いて実施例 1〜 3のアルデヒド基結合アミノアルキルシラ ンを導入した本発明基板は、 DN A固定化効率及びハイブリダィズ効率が高く、 かつ基板自身による蛍光が抑制されていた。

The substrate of the present invention in which the aldehyde group-bonded aminoalkylsilane of Examples 1 to 3 was introduced using the substrate of Reference Example 1 had high DNA fixing efficiency and hybridization efficiency, and suppressed fluorescence by the substrate itself. .

 (Reference example 2)

Mixing 50% by weight of stabilizer wax with 50% by weight of carbon black to produce a pellet containing a high concentration of black pigment. 5% by weight of the aforementioned pellets were blended with 95% by weight of the hydrogenated random copolymer to prepare a saturated cyclic polyolefin resin colored black. Then, a slide plate having a thickness of one thigh was injection-molded using this resin.

 (Reference example 3)

 5% by weight of the pellet described in Reference Example 2 was blended with 95% by weight of polystyrene to produce black-colored polystyrene. Then, a slide plate having a thickness of 1 mm was injection-molded using this resin.

 (Comparative Reference Example 2)

 A one-thick slide plate was injection molded using a random copolymer of ethylene and dicyclopentadiene.

 (Comparative Reference Example 3)

 A slide blade with a thickness of 1 lM was injection molded using polystyrene.

 (Comparative Reference Example 4)

 A quartz glass slide glass (S111) manufactured by Matsunami Glass Co., Ltd. was used. (Refer to Reference Examples 2 and 3 and Comparative Reference Examples 2, 3 and 4 with the sample を slide on which no sample was placed, and use an epi-fluorescence microscope (excitation light wavelength 532 nm, fluorescence wavelength 560 The fluorescence was photographed with a CCD camera and the amount of fluorescence was compared and evaluated from the image data.

 Table 7 shows the evaluation results. From this, it was confirmed that the background fluorescence was suppressed by performing the opacity treatment with the pigment. In particular, it was confirmed that the background value of Reference Example 2 was lower than that of the quartz glass of Comparative Reference Example 4.

 Table 7

 Fluorescence intensity (quartz glass = 100) Reference Example 2 Black-colored saturated cyclic polyolefin resin 62

Reference Example 3 Black colored polystyrene 105

Comparative Reference Example 2 Saturated cyclic polyolefin resin 262

Comparative Reference Example 3 Polystyrene 1549

Comparative Reference Example 4 Quartz glass 100 The substrate of the present invention into which the aldehyde group-bonded aminoalkylsilane was introduced in the same manner as in Examples 1 to 4 using the resins of Reference Examples 2 and 3, particularly the resin of Reference Example 3, had high DNA binding efficiency and high hybridization efficiency. The substrate had a low fluorescent pack ground.

 (Reference Example 4)

 Using the same norbornene-based polyolefin resin as in Example 1, the substrate shown in FIG. 1 having a length of 76 mm, a width of 26 mm, a thickness of the sample fixing portion of 0.9 mm, and a thickness of the outer edge of one thigh was injection-molded. . The surface roughness of the molded product was 0.002 to 0.003 zm, and no distortion or warpage was observed over the entire substrate. The substrate was scanned using a microarray scanner "ScanArray LITE" manufactured by Packard BioChip Technologies. Scanning conditions were 90% laser output and 90% PMT sensitivity. Fig 5

As shown in the histogram (top), the amount of background fluorescence was less uneven, indicating that there was no deformation such as warpage or distortion on the substrate surface.

 (Comparative Reference Example 5)

 Using the same resin as in Reference Example 4, a 76 x 26 x 1 mm slide glass substrate was injection molded. The surface roughness of the molded product was 0.002 to 0.003 ^ m, and no distortion or warpage was observed over the entire substrate. As a result of scanning under the same conditions as in Reference Example 4, unevenness was observed in the amount of background fluorescence, as shown in the histogram of FIG. 5 (lower). This is because the substrate surface was deformed by the stress from the fixing jig.

The substrate of the present invention into which the aldehyde-bonded aminoalkylsilane was introduced in the same manner as in Examples 1 to 3 using the injection-molded product of Reference Example 4 had a high DNA binding efficiency and a high pre-hydidization efficiency, and also required a The sample fixed surface was not deformed due to uneven force application. Industrial applicability

 The microchip substrate of the present invention has a high DNA immobilization efficiency and a uniform amount of DNA immobilized on the substrate when immobilizing DNA by spotting DNA. It is suitable as a DNA chip substrate because of its high sensitivity and small variation in spot detection intensity in detection.

Claims

The scope of the claims 1. A plastic substrate for microchips, characterized in that an aminoalkylsilane in which an aldehyde group derived from dartartaldehyde is introduced into an amino group is present on the surface.  2. The method according to claim 1, wherein the aminoalkylsilane is present on the surface. 3. The method according to claim 1 or 2, wherein the alkylsilane is present on the surface. 4. The plastic substrate for a microchip according to claim 3, wherein the molecular chain length of the alkylsilane is shorter than that of the aminoalkylsilane.  5. The plastic substrate for a microchip according to any one of claims 1 to 4, wherein the plastic is a saturated cyclic polyolefin.  6. The microchip plastic substrate according to claim 5, wherein the saturated cyclic polyolefin-based resin contains at least one of norpolene or a norpolene derivative as a monomer unit.  7. The microchip plastic substrate according to any one of claims 1 to 6, wherein the plastic contains a pigment and is opaque.  8. The plastic substrate for a microchip according to claim 7, wherein the pigment is a black pigment.  9. The plastic substrate for a microchip according to claim 7, wherein the pigment is a white pigment.  10. The pigment according to any one of claims 7 to 9, wherein the content of the pigment is 1 to 60% by weight. 11. The method according to any one of claims 1 to 10, wherein a means for limiting a contact surface with the measuring device is provided on the back surface of the substrate. 12. The microchip plastic substrate according to claim 11, wherein the means for limiting the contact surface is to provide a projection on a part of the back surface of the sample fixing surface of the substrate.  13. The mask according to claim 12, wherein the convex portion is an outer edge portion that is thicker than a thickness of the sample fixing surface. 14. On plastic substrates  (1) Surface oxidation treatment,  (2) contact with a solution containing aminoalkylsilane, and  (3) contact with a solution containing daltaraldehyde, A method for producing a plastic substrate for microchips, characterized in that an amino group in which an aldehyde group derived from dartartaldehyde has been introduced into an amino group is present on the substrate surface by a step comprising:  15. The method for producing a plastic substrate for microchips according to claim 14, wherein a hydroxyl group is introduced into the surface of the plastic substrate by acid treatment of the surface.  16. The method according to claim 14 or 15, wherein the surface oxidation treatment is a low-temperature plasma treatment.  17. The method for producing a microchip plastic substrate according to claim 16, wherein the low-temperature plasma treatment is performed in an oxygen or gas atmosphere containing oxygen.  18. The method for producing a plastic substrate for a microchip according to any one of claims 15 to 17, wherein a hydroxyl group is introduced into a radical state or a 7T-bonded carbon atom immediately after the oxidation treatment.  19. The method according to claim 18, wherein the method of introducing a hydroxyl group into a radical state or a C-bonded carbon atom immediately after the oxidation treatment is to contact a water molecule.  20. The method for producing a microchip plastic substrate according to any one of claims 14 to 19, wherein the plastic is a saturated cyclic polyolefin. 21. Saturated cyclic polyolefin resin 21. The plastic substrate for a microphone opening chip according to claim 20, wherein at least one of the above is included as one monomer unit.  22.7 The carbon atoms present in the molecular layer constituting the surface of the substrate by introducing a hydroxyl group into the carbon atoms bonded to the 2.7t bond, wherein the carbon atoms having a 7T bond are 15% or less. 20. The method for producing a microchip plastic substrate according to 20 or 21.  23. The method for producing a plastic substrate for microchips according to any one of claims 14 to 22, wherein the solution containing aminoalkylsilane contains alkylsilane.  22. The method for producing a plastic substrate for a microchip according to claim 22, wherein the molecular chain length of the alkylsilane is shorter than that of the aminoalkylsilane.  25. A DNA chain having an amino group introduced into its terminal is dissolved in a solution, and this solution is brought into contact with the microchip substrate according to any one of claims 1 to 13 to obtain an amino group of a DNA chain. A method for using a plastic substrate for microchips, wherein a DNA chain is fixed on a substrate by covalently bonding the DNA chain to an aldehyde group introduced on the substrate.