JP3786248B2 - Chemically modified substrate and method for producing the same - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a substrate having a chemical modification capable of immobilizing a nucleic acid or protein, which is useful in the fields of molecular biology and biochemistry. For example, DNA is immobilized on the surface of a solid support, stored, taken out when necessary, and a DNA segment is amplified by polymerase chain reaction (PCR) to analyze the DNA. It was achieved with
[0002]
[Prior art]
Genetic analysis is useful in the fields of molecular biology and biochemistry, and in recent years it has also been used in medical fields such as disease detection.
[0003]
In gene analysis, DNA chips have recently been developed and the analysis speed has been significantly increased. However, the conventional DNA chip is a method in which a polymer such as polylysine is applied to the surface of a slide glass or silicon substrate, and then DNA is fixed. In addition, a method of synthesizing oligonucleotides on a glass substrate using a semiconductor technique such as photolithography is used.
[0004]
However, in the method of immobilizing DNA by applying a polymer such as polylysine on the surface of a slide glass or silicon substrate, the DNA immobilization state is unstable, and the DNA is peeled off in the hybrid formation process or washing process. Occurs. In addition, a DNA chip using semiconductor technology has a problem that it is very expensive due to the complexity of the manufacturing process.
In order to solve such problems, it is necessary to immobilize DNA on the solid support surface with high density and strength.
[0005]
Conventionally, a substrate obtained by chemically modifying the surface of a solid support is known. However, in order to bind DNA to a carboxylic acid or the like, activation of the carboxylic acid is required, so that chemical modification of the substrate alone is not sufficient.
[0006]
An object of the present invention is to provide a substrate that can efficiently perform elucidation of DNA and DNA storage, and that has been subjected to chemical modification useful in the fields of molecular biology, biochemistry, and the like. . Another object of the present invention is to provide a substrate for stably immobilizing DNA or protein.
[0007]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have a hydrocarbon group containing an activated ester group such as N-hydroxysuccinimide ester or p-nitrophenol ester by chemically modifying the surface of the solid support. The inventors have found that the substrate can stably fix DNA and the like, and have reached the present invention.
That is,
Method for producing a substrate which has been subjected to chemical modification according to claim 1, the solid support surface chlorination and then amination to form a primary amino group in the solid support surface, said primary amino group And a dicarboxylic acid obtained by reacting dicarboxylic acid with carbodiimide or dicyclohexylcarbodiimide and p-nitrophenol, and reacting with p-nitrophenol activated esterified dicarboxylic acid, N-hydroxyl at one end The other end of the hydrocarbon group to which an activated ester group consisting of a succinimide ester group or a p-nitrophenol ester group is bonded is fixed to the surface of the solid support through an amide bond.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The substrate of the present invention is characterized in that a specific chemical modification is applied to the surface of a solid support. The chemical modification in the present invention means immobilization of a group having an active ester group bonded to the end of a hydrocarbon group on the surface of a solid support through an amide bond. Such chemical modification makes it easier to fix DNA or protein on the surface of the substrate.
[0009]
The hydrocarbon group is preferably those having 0 to 12 carbon atoms, especially 0 to 6 carbon atoms.
Examples thereof include monocarboxylic acids such as formic acid, acetic acid, and propionic acid; dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid; and polyvalent carboxylic acids such as trimellitic acid. Of these, oxalic acid and succinic acid are preferred.
[0010]
As the active ester group bonded to the terminal of the hydrocarbon group, N-hydroxysuccinimide ester or p-nitrophenol ester is preferable.
[0011]
Such chemical modification is to immobilize a group in which an active ester group is bonded to the end of a hydrocarbon group via an amide bond on a solid support.
For example, when the active ester group is an N-hydroxysuccinimide ester group, the solid support is irradiated with ultraviolet light in chlorine gas to chlorinate the surface, then irradiated with ultraviolet light in ammonia gas and then aminated, and then an appropriate acid is used. Carboxylation is performed using chloride, and the terminal carboxyl group can be dehydrated and condensed with carbodiimide or dicyclohexylcarbodiimide and N-hydroxysuccinimide.
An example of the dehydration condensation when this method is adopted will be described. The surface is chemically modified, and the solid support having a carboxyl group is dissolved in carbodiimide or dicyclohexylcarbodiimide, and N-hydroxysuccinimide or p-nitrophenol. It is immersed in the 1,4-dioxane solution, washed and dried. In this way, a substrate to which a hydrocarbon group having a terminal N-succinimide ester group or p-nitrophenol ester group is bonded is obtained.
[0012]
Further, as a more preferable method than the above method, it is particularly formed by dehydrating condensation of one ester group of the activated diester to a primary amino group previously formed on a fixed support (substrate) such as diamond. It is desirable.
[0013]
An activated diester refers to one having two active ester groups as described above. The ester groups are preferably located at both ends in the activated diester, preferably those having 0 to 12 carbon atoms, preferably 0 to 6 carbon atoms. The skeleton portion excluding the ester group is preferably a linear saturated fatty acid.
[0014]
As a method using an activated diester, chlorination is carried out in the same manner, and after the amination, an activated ester obtained by N-hydroxysuccinimide activated esterification of oxalic acid (dicarboxylic acid) in advance is reacted with this amino group. And a method for obtaining a desired substrate.
[0015]
In this method, N-hydroxysuccinimide activated esterification refers to a 1,4-dioxane solution in which 2.5 mg / ml of carbodiimide and 1.5 mg / ml of N-hydroxysuccinimide are dissolved (for one 3 mm square diamond). In an amount of 100 μl) and reacted for 15 minutes to obtain an activated diester. The activated ester is attached with an amino group and bound to a solid support to complete the chemical modification.
This method was first developed by the present inventors. By using a product in which the surface of the solid support is chemically modified with an activated ester group, the user can easily immobilize DNA directly on the surface of the solid support by an amide bond.
[0016]
Although oxalic acid dichloride is difficult to obtain, the solid support surface can be easily chemically modified by converting oxalic acid into an N-hydroxysuccinimide activated ester.
[0017]
In the present invention, the solid support to be chemically modified as described above includes metals such as diamond, gold, silver, copper, aluminum, tungsten and molybdenum; laminates of the above metals and ceramics; plastics such as polycarbonate and fluororesin Etc.
Other materials can be used as long as they are chemically stable, and examples thereof include graphite and diamond-like carbon. Also, a mixture of plastic and the above metals, ceramics, diamond or the like may be used.
[0018]
Of these, diamond is preferred from the viewpoint of thermal conductivity. Since diamond is excellent in thermal conductivity and can be rapidly cooled, the heat cycle time for repeating heating and cooling such as PCR can be effectively shortened.
The thermal conductivity of the substrate of the present invention is 0.1 W / cm · K or more, preferably 0.5 W / cm · K or more, particularly preferably 1.0 W / cm · K or more. This is because, by setting the DNA to 1.0 W / cm · K or more, when performing PCR or the like by immobilizing DNA on the chemically modified portion of the substrate of the present invention, the followability of heating and cooling is excellent.
[0019]
As a material for the diamond substrate, any of synthetic diamond, high pressure formed diamond, natural diamond, and the like can be used. Further, the structure may be either a single crystal or a polycrystal. From the viewpoint of productivity, it is preferable to use diamond manufactured using a vapor phase synthesis method such as a microwave plasma CVD method.
[0020]
The method for forming a substrate of the present invention can be performed by a known method. For example, a microwave plasma CVD method, an ECRCVD method, an IPC method, a direct current sputtering method, an ECR sputtering method, an ion plating method, an arc ion plating method, an EB vapor deposition method, a resistance heating vapor deposition method, and the like can be given. Moreover, what formed resin by mixing resin as a binder with metal powder, ceramic powder, etc. is mentioned. In addition, a material obtained by compacting a raw material such as a metal powder or a ceramic powder using a press molding machine and sintering at a high temperature is also included.
[0021]
The substrate surface of the substrate of the present invention is desirably intentionally roughened. This is because such a roughened surface is convenient for fixing a large amount of DNA or the like by increasing the surface area of the substrate. The shape of the substrate is not particularly limited, such as a flat plate shape, a thread shape, a spherical shape, a polygonal shape, and a powder shape.
Further, the diamond substrate may be a composite of diamond and another substance (for example, a substrate having two layers).
[0022]
【Example】
The following examples illustrate the invention.
Example 1
The surface was chlorinated by irradiating 3 mm square CVD diamond in chlorine gas with ultraviolet rays and then aminated by irradiation with ultraviolet rays in ammonia gas, and then refluxed in chloroform with acid chloride for carboxylation.
This surface-modified solid support was placed in a 1,4-dioxane solution (100 μl per 3 mm square diamond) in which carbodiimide 2.5 mg / ml and N-hydroxysuccinimide 1.5 mg / ml were dissolved. And the terminal carboxyl group was dehydrated and condensed. After the completion of the reaction, the substrate was washed with water, further washed with a 1,4-dioxane solution and dried to obtain a chemically modified substrate.
[0023]
Example 2
The surface of the silicon substrate is chlorinated by irradiating a 3 mm square silicon substrate in chlorine gas and then aminated by irradiating with ultraviolet light in ammonia gas, and oxalic acid (dicarboxylic acid) is previously added to this amino group with N-hydroxysuccinimide. The activated diester obtained by activated esterification was reacted.
N-hydroxysuccinimide activated esterification was performed as follows. Oxalic acid was dissolved in 1,4-dioxane solution (100 μl per 3 mm square diamond) dissolved in carbodiimide 2.5 mg / ml and N-hydroxysuccinimide 1.5 mg / ml, and reacted for 15 minutes. An activated diester was obtained. The activated diester was bonded to a solid support previously provided with an amino group to obtain a chemically modified substrate.
[0024]
After immobilizing a 20-mer oligonucleotide using the substrates prepared in Example 1 and Example 2 as described above, hybridizing with a fluorescently labeled probe having a complementary sequence, and immobilizing the oligonucleotide using a fluorometer The amount of conversion was estimated. As a result, in Example 1, it was 38 pmol per 3 mm square substrate, and in Example 2, it was 35 pmol per 3 mm square substrate. It was revealed that both oligonucleotides were fixed at high density.
[0025]
【The invention's effect】
Since the chemically modified substrate of the present invention is chemically modified and has an activated ester group, nucleic acids such as DNA can be stably immobilized, which is advantageous for PCR. Moreover, the production method of the present invention can efficiently produce a substrate capable of stably immobilizing DNA or the like. In particular, by using a diester obtained by activating N-hydroxysuccinimide using carbodiimide and N-hydroxysuccinimide, an activated ester group is formed on the surface of the solid support without refluxing in chloroform in the presence of acid chloride. Can be chemically modified. In addition, by using the product in this state, the user can easily fix the DNA.

Claims (1)

The surface of the solid support is chlorinated and then aminated to form a primary amino group on the surface of the solid support. The primary amino group includes dicarboxylic acid, carbodiimide or dicyclohexylcarbodiimide, and N-hydroxysuccinimide or p. -N-hydroxysuccinimide activated esterified dicarboxylic acid or p-nitrophenol activated esterified dicarboxylic acid obtained by reacting nitrophenol is reacted with N-hydroxy at one end A chemical modification characterized by immobilizing the other end of a hydrocarbon group to which an activated ester group comprising a succinimide ester group or a p-nitrophenol ester group is bonded to the surface of a solid support through an amide bond A method for producing the applied substrate.