JP2003009859A - Substrate for immobilizing nucleic acid, method for producing the substrate, and glass plate, and method for analyzing nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a substrate enabling a nucleic acid to be stably immobilized on a specified area of the surface thereof. SOLUTION: This substrate has the following structure: a photocatalyst layer 2 having photocatalytic activity (e.g. TiO2 film) is provided on the surface of a glass substrate 1 and the surface of the photocatalyst layer 2 is laminated with a thin film 3 of a water-repellent group-bearing organic compound splittable by photocatalytic action (e.g. fluoroalkyl group-bearing silane compound); wherein the thin film 3 has an arrangement pattern where 1st areas 3b and 2nd areas 3a showing relatively greater wetting tendency and less wetting tendency respectively on irradiation with light via a photomask are regularly arranged, and compound films 4 for immobilizing a nucleic acid are provided only on the surface of the 1st areas 3b; thus, the nucleic acid can be immobilized stably on the films 4.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nucleic acid immobilizing substrate, a method for producing a nucleic acid immobilizing substrate, a glass plate, and a nucleic acid analyzing method. , A substrate for nucleic acid immobilization that can be used for nucleic acid analysis such as monitoring of expression of genomic genes, a method for producing the same, a glass plate for immobilizing nucleic acid on the nucleic acid immobilization substrate, and a glass plate immobilized on the glass plate The present invention relates to a nucleic acid analysis method for analyzing a nucleic acid.

[0002]

In the field of genetic engineering, nucleotide sequencing,
DNA (deoxyribonucleic acid) and RN are used for diagnosis of infectious diseases and genetic diseases, and also for monitoring expression of genomic genes.
A (ribonucleic acid) or a nucleic acid formed by a mixture thereof is immobilized on a substrate to prepare a nucleic acid array or a nucleic acid chip such as a so-called DNA array or DNA chip, and hybridization is performed using the nucleic acid array or the nucleic acid chip. Gene analysis is widely performed. For example, a hybrid nucleotide sequencing method is expected to be put to practical use as a high-speed and low-cost method (SBH (sequencing by
hybridization; R. Drmanac et al. , Science, 260, 16
49 (1993)), and a method for monitoring gene expression patterns using a DNA array (M. Schena et al., Sci.
ence, 270, 467-470 (1995)) has attracted attention as a method for rapidly analyzing the expression of a large amount of genes.

As a substrate material for immobilizing nucleic acid such as DNA on a substrate, a resin substrate, a glass substrate, a metal substrate or the like can be generally used.
When the detection of hybridization is performed by using a fluorescent substance without using a radioactive isotope, a glass substrate or a silicon substrate containing no fluorescent substance is considered to be suitable.

However, when a glass substrate is used as a substrate material for a nucleic acid immobilization substrate, the glass substrate is formed of an inorganic material containing a silicon compound as a main component,
It does not have the functional group necessary for immobilizing nucleic acid on the surface of the glass substrate. Therefore, in order to immobilize the nucleic acid on the surface of the glass substrate, a compound film for immobilizing nucleic acid containing a predetermined functional group is used. It must be formed on the surface of the substrate.

As the predetermined functional group, an amino group or a carboxyl group is preferable. Conventionally, aminosilane containing an amino group is used as a nucleic acid fixing compound, and the aminosilane is coated on the surface of a glass substrate. (Z. Guo et al., Nucl. Acids. Res., 2
2, p.5456-p. 5465 (1994)), a method of using polylysine containing an amino group and a carboxyl group, and coating the surface of a glass substrate with the polylysine (M. Schena
et al. , Science, 270, 467-470 (1995)).

[0006]

However, in the above-mentioned prior art, the nucleic acid-immobilizing compound such as aminosilane or polylysine is coated on the surface of the glass substrate and then the nucleic acid is immobilized on the nucleic acid-immobilizing compound film using a jig or the like. Since the nucleic acid is immobilized, an operation error or the like occurs during the work of immobilizing the nucleic acid, and the nucleic acid adheres to the outside of the predetermined area on the glass substrate, or the nucleic acid is dropped in the predetermined area, but There is a risk that the particles may scatter and adhere to the outside of a predetermined area to be immobilized, which may make it difficult to produce a desired nucleic acid array or nucleic acid chip.

The present invention has been made in view of the above problems, and it is a substrate for nucleic acid immobilization capable of stably immobilizing nucleic acids in a predetermined region on the surface of a substrate, a method for producing a substrate for nucleic acid immobilization, Provided are a glass plate capable of reliably immobilizing nucleic acid in a predetermined region using the nucleic acid immobilizing substrate, and a nucleic acid analyzing method for analyzing the nucleic acid immobilized on the nucleic acid immobilizing substrate. The purpose is to

[0008]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to make it possible to form a nucleic acid-immobilizing compound film for stably immobilizing nucleic acids on a glass substrate only in a desired region on the glass substrate. However, there is a correlation between the adherence of the nucleic acid-immobilizing compound to the glass substrate and the wettability on the glass substrate,
Nucleic acid immobilizing compounds easily attach to highly wettable parts,
We have found that it is difficult to adhere to parts with low wettability.

The present invention has been made on the basis of such findings, and the nucleic acid-immobilizing substrate according to the present invention has a first region having large wettability and a second region having small wettability regularly. It is characterized in that the array pattern arrayed in is formed on the surface of the glass substrate.

That is, according to the above structure, the nucleic acid-immobilizing compound film is prevented from adhering to the second region having low wettability, and the nucleic acid-immobilizing compound film is provided only in the first region having high wettability. It can be formed, and the nucleic acid can be stably immobilized only in a desired region on the glass substrate.

As the nucleic acid fixing compound, an organic compound containing an amino group or a carboxyl group is preferable.

That is, in the nucleic acid immobilizing substrate of the present invention, a nucleic acid immobilizing compound film containing at least one functional group of an amino group and a carboxyl group is formed on the surface of the first region. It is also preferable that the nucleic acid is characterized in that it is possible to easily stably immobilize the nucleic acid only in the first region by providing such a constitution.

Then, the present inventor has conducted earnest research for effectively differentiating the wettability of the glass substrate surface. As a result, a photocatalyst layer made of a photoactive substance such as titanium oxide is formed on the glass substrate surface. An organic compound that decomposes by photocatalysis is laminated on the photocatalyst layer, and then the organic compound film is irradiated with light through a photomask having a predetermined pattern, so that the exposed portion is decomposed by photocatalysis and gets wet. It was found that the wettability is significantly different from that of the non-exposed area as a result.

Therefore, in the nucleic acid immobilizing substrate of the present invention, a photocatalytic layer made of a photoactive substance is formed on the surface of the glass substrate, and a thin film made of an organic compound which is decomposed by a photocatalytic action is laminated on the photocatalytic layer. The first region and the second region are patterned and formed on the thin film.

In addition, the method for producing a substrate for immobilizing nucleic acid according to the present invention is a thin film made of an organic compound, which has a photocatalytic layer having photoactivity formed on the surface of a glass substrate and is decomposed by photocatalysis to change wettability. And irradiating the thin film with light through a photomask having a predetermined pattern to form a pattern in which a first region having high wettability and a second region having low wettability are regularly arranged. Is characterized by.

Then, the present inventor has conducted further diligent research and found that a mixed film in which a photoactive substance and an organic compound which is decomposed by a photocatalytic action are mixed is formed on a glass substrate, and the above-mentioned photomask is used to form the mixed film. Even when the mixed film is irradiated with light, the exposed thin film portion is decomposed by the photocatalytic action to increase the wettability as a result, and as a result, the difference in wettability between the non-exposed portion is remarkable. I got the knowledge that

Therefore, in the nucleic acid immobilizing substrate of the present invention, a mixed film in which a photoactive substance and an organic compound which is decomposed by photocatalysis are mixed is formed on the surface of the glass substrate, and the first film is formed.
It is also preferable that the region and the second region are formed by patterning on the mixed film,
Further, the method for producing a substrate for immobilizing nucleic acid of the present invention, after forming a mixed film containing a photocatalytic substance having photoactivity and an organic compound that decomposes by photocatalytic action and changes wettability on the surface of a glass substrate, The mixed film is irradiated with light through a photomask having a predetermined pattern to form a pattern in which first regions having high wettability and second regions having low wettability are regularly arranged. It is also preferable to

As the organic compound, it is preferable to use a compound having a water repellent group, particularly a fluoroalkyltrialkoxysilane, a hydrolyzate thereof, or a polycondensate thereof, and as the photoactive substance. It is preferable to use titanium oxide which is particularly excellent in photocatalysis.

Further, the glass plate according to the present invention is characterized in that the nucleic acid is immobilized on the first region formed on the nucleic acid immobilizing substrate.

According to the above glass plate, since the nucleic acid-immobilizing compound film is formed only in the first region having high wettability, it becomes possible to stably immobilize the nucleic acid only in the first region. A nucleic acid array or nucleic acid chip having a desired regular array pattern can be easily obtained.

The nucleic acid analysis method according to the present invention is characterized in that the nucleic acid immobilized on the nucleic acid-immobilizing substrate of the glass plate is analyzed.

According to the above-mentioned analysis method, a nucleic acid sample regularly arranged on a glass plate can be analyzed, so that various gene analyzes such as nucleotide sequence determination, infectious diseases and genetic diseases can be efficiently carried out. It becomes possible to do.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, embodiments of the present invention will be described in detail with reference to the drawings.

FIG. 1 is a sectional view schematically showing an embodiment of a nucleic acid immobilizing substrate according to the present invention, in which the surface of a non-alkali glass substrate 1 made of aluminoborosilicate or the like is provided. A photocatalyst layer 2 having photocatalytic activity is formed, and a thin film 3 made of an organic compound which is decomposed by a photocatalytic action is laminated on the surface of the photocatalyst layer 2. And the thin film 3 is
The first region 3b having a relatively high wettability and the second region 3a having a low wettability have an array pattern regularly arranged, and the nucleic acid is present on the surface of the first region 3b. The fixing compound film 4 is formed.

The photocatalyst layer 2 is composed of TiO ₂ , ZnO, WO ₃ ,
Fe ₂ O ₃ , SrTiO ₃ , In ₂ O ₃ , MoO ₂ , TiO ₂
Is formed of a light catalytically active material such as -pt-RuO _2, a thick film is formed on 10 nm to 200 nm.

The thin film 3 is composed of an organic compound which is decomposed by a photocatalytic action by light irradiation to change its wettability, for example, an organic compound having a water-repellent group, and the thin film 3 has a thickness of 1 n.
It is formed to have a thickness of m to 100 nm, preferably 1 nm to 50 nm. In addition, the first area 3b and the second area 3a are 1
Square with sides of 10 μm to 500 μm or diameter of 10 μm
It is formed by patterning in a grid pattern in a desired shape such as a circle of m to 500 μm, an ellipse or a regular hexagon.

As the organic compound forming the thin film 3, one or more alkyl groups (C _n H _{2n + 1} ) (n
Is an integer of 1 or more; hereinafter the same ") and a silane compound containing a fluoroalkyl group (C _n F _{2n + 1} (CH ₂ ) ₂ ) can be used.

Specifically, a silane containing an alkyl group
As the compound, a compound represented by the general formula C_nH_{2n + 1}SiCl₃, (C_nH
_{2n + 1})₂SiCl₂, (C_nH_{2n + 1})₃Represented by SiCl
Chlorosilane compound, general formula C_nH_{2n + 1}Si (OCH₃)
₃, (C_nH_{2n + 1})₂Si (OCH₃)₂, (C_nH_{2n + 1})₃
SiOCH₃, C_nH_{2n + 1}Si (OC₂H_Five)₃, (C_nH
_{2n + 1})₂Si (OC₂H_Five)₂, (C_nH_{2n + 1})₃SiOC₂
H_FiveAn alkoxysilane compound represented by the general formula C_nH
_{2n + 1}Si (OCOCH₃)₃, (C_nH_{2n + 1})₂Si (OC
OCH₃)₂, (C_nH_{2n + 1})₃SiOCOCH₃Indicated by
Acyloxysilane compound, general formula C_nH_{2n + 1}Si (N
CO)₃Or (C_nH_{2n + 1})₂Si (NCO)₂, (C_nH
_{2n + 1})₃Isocyanate silanization represented by SiNCO
We can mention compound.

A sila containing a fluoroalkyl group
The compound of general formula C_nF_{2n + 1}(CH₂)₂SiC
l₃A trichlorosilane compound represented by the general formula C_nF
_{2n + 1}(CH₂)₂Si (OCH₃)₃, C_nF_{2n + 1}(CH₂)
₂Si (OCH₂CH₃)₃Trialkoxy sila
Compounds, general formula C_nF_{2n + 1}(CH₂)₂Si (OCOC
H₃)₃A triacyloxysilane compound represented by the general formula
C_nF_{2n + 1}(CH₂)₂Si (NCO)₃Torii indicated by
There may be mentioned cyanate silane compounds.
For silane compounds containing fluoroalkyl groups,
Fluoroalkyltrimetho having 13 to 22 elementary atoms
Use xysilane or fluoroalkyltriethoxysilane.
It is preferably used.

As the nucleic acid immobilizing compound, an aminosilane compound containing an amino group and polylysine ((NH ₂ (CH ₂ )) containing both an amino group and a carboxyl group are used.
Polymers such as ₄ CH (NH ₂ ) COOH) _n ) can be used.

Next, a method of manufacturing the above-mentioned substrate for immobilizing nucleic acid will be described.

FIG. 2 is a manufacturing process diagram showing a method for manufacturing a nucleic acid immobilizing substrate according to the present invention.

First, as shown in FIG. 2A, the photocatalyst layer 2 is formed on the surface of the glass substrate 1. That is, using an oxide such as TiO ₂ or ZnO exhibiting photocatalytic activity, a sol-gel method, a vacuum deposition method, a chemical vapor deposition method (CVD method),
The photocatalyst layer 2 having a film thickness of 10 nm to 100 nm is formed by a thin film forming method such as a sputtering method or a fine particle baking method. For example, when the photocatalyst layer 2 made of TiO ₂ is formed by the sol-gel method, titanium alkoxide or titanium acetylacetonate in which alcohol is bound to titanium,
An organic titanium compound solution such as titanium carboxylate is applied to the surface of the glass substrate 1 by a dip coating method, a spin coating method, a spray coating method or the like, and TiO ₂ fine particles are deposited to form a TiO ₂ sol. And then 10
Heat treatment is performed at a heating temperature of 450 ° C. to 550 ° C. for a period of 2 minutes to 2 hours to gelate the TiO ₂ sol, which results in TiO ₂ sol.
To form a photocatalyst layer 2.

Further, commercially available photocatalytically active fine particles such as T
A dispersion sol in which fine particles of iO ₂ are dispersed in a binder such as water or ethyl alcohol is prepared, and the dispersion sol is used as a glass substrate 1.
The photocatalyst layer 2 can also be easily formed on the surface of the glass substrate 1 by applying a heat treatment under substantially the same conditions as described above after coating the surface of the glass substrate 1.

Next, as shown in FIG. 2B, an organic compound that decomposes by photocatalysis and changes in wettability, for example, an organic compound containing a water-repellent functional group such as fluorotrialkoxysilane is used. , Sol-gel method, vacuum deposition method, C
A thin film 3 made of the organic compound is formed on the photocatalyst layer 2 by the VD method or the like.

Next, as shown in FIG. 2C, the surface of the thin film 3 is covered with a photomask 5 having a large number of holes 5a of a predetermined shape, and ultraviolet rays are irradiated from above as shown by an arrow A. The hole 5a of the photomask 5 is formed in a shape that matches the bottom shape of the nucleic acid immobilization region to be formed.

In this way, the exposed portion is decomposed by the photocatalytic action, and as a result, the wettability in the exposed portion is increased. That is, the thin film 3 is divided into a light transmitting region and a light blocking region by the photomask 5, and the thin film portion corresponding to the light transmitting region is exposed and decomposed by photocatalysis, and the organic compound forming the thin film is water repellent. As a result, the thin film 3 loses its wettability, and as a result, the thin film 3 is divided into a first region 3b having a relatively high wettability and a second region 3a having a low wettability. For example, when the thin film 3 is formed of a fluoroalkyl group-containing alkoxysilane or a hydrolyzate or polycondensation product thereof, the non-exposed portion does not lose water repellency, but the wettability does not change. In the exposed area, the fluoroalkyl group decomposes due to the action of the photocatalyst to lose the water repellency and increase in size, and a regular array pattern having the first region 3b and the second region 3a is formed on the thin film 3. It

By this light irradiation, the photoactivity of the photocatalyst layer is expressed to form a pattern structure in which regions having different surface energies are periodically present in the exposed and unexposed regions. The light used for irradiation may be any light as long as the photocatalytically active substance in the photocatalytic layer exhibits photocatalytic activity, and it is preferable to use ultraviolet rays having an excitation wavelength of 380 nm or less.

Next, as shown in FIG. 2 (e), a nucleic acid such as an aminosilane compound containing a functional group suitable for immobilizing a nucleic acid, eg, an amino group, or polylysine containing an amino group and a carboxyl group is immobilized. The compound for use is formed on the surface of the first region 1a having high wettability. As the coating forming method, a casting method, a dip coating method, a spin coating method, a gravure coating method, a flexible coating method, a roll coating method, a spray coating method, or the like can be used.

In particular, in the spray coating method, since the nucleic acid-immobilizing compound solution adheres to the substrate in the form of droplets, even if the compound solution for nucleic acid fixation adheres to the second region 3a having a small wettability, the first solution having a large wettability is used.
Therefore, when the spin coating method is used, the nucleic acid-immobilizing compound is preferably applied only to the first region 3b having high wettability.

Then, moisture is removed by appropriately combining heat treatment, natural drying treatment, etc., whereby the nucleic acid fixing compound film 4 is formed only on the first region 3b on the glass substrate 1.
A nucleic acid-immobilized substrate coated with is obtained.

By immobilizing the nucleic acid 6 on the surface of the first region 3b of the thus obtained substrate for nucleic acid immobilization as shown in FIG. 3, the nucleic acid 6 having a predetermined pattern is regularly arrayed. The manufactured glass plate is manufactured.

The nucleic acid 6 to be immobilized may be either DNA or RNA, the molecular weight of the nucleic acid 6 is not particularly limited, and it may be a synthetic oligonucleotide or a natural nucleic acid, and a PCR product or the like is used. May be.

When the analysis of the sample nucleic acid to be hybridized with the nucleic acid (including the oligonucleotide) immobilized on the glass plate for the purpose of analyzing the nucleotide sequence is carried out by hybridization, the sample nucleic acid or the glass plate is immobilized. It is preferable that any one of the nucleic acids thus modified is pre-labeled. The labeling method is not particularly limited, and for example, there is a method using a radioisotope or a fluorescent dye. The result of hybridization is
Although it can be measured by a method compatible with various labeling methods, in the gene expression monitoring method, by labeling the sample nucleic acid with a plurality of fluorescent dyes having different detection wavelengths, the expression intensity between the sample nucleic acids can be parallelized. It is preferable to use a label obtained by a fluorescence method using a fluorescent dye because it can be detected.

The immobilization of the nucleic acid 6 on the nucleic acid immobilizing substrate is carried out by dropping an appropriate amount of the nucleic acid solution onto the nucleic acid immobilizing compound film 4.
It can be carried out by drying, irradiation with ultraviolet rays, or a combination of both, but irradiation with ultraviolet rays is preferable for stable immobilization. Further, in consideration of the analysis of nucleic acid, it is preferable to carry out a post-treatment using various acids, hot water, alcohol or the like in order to remove the nucleic acid and the like not immobilized on the substrate for immobilizing nucleic acid. Methyl-2
It is more preferred to use a post-treatment using an SMP solution dissolved in a pyrrolidone solution and added with a predetermined amount of sodium borate solution.

It should be noted that these optimum conditions can be determined by appropriately changing the conditions based on the measurement results by various gene analysis methods.

The glass plate on which the nucleic acid is immobilized in this manner is used when performing various gene analyzes as a nucleic acid array or a nucleic acid chip.

Other methods for producing the nucleic acid array include a method of directly synthesizing an oligonucleotide on a nucleic acid-immobilizing substrate, a synthesized oligonucleotide, or
A method of immobilizing a PCR product or the like on a nucleic acid immobilizing substrate may be used.

The thus-prepared glass plate has a stable nucleic acid immobilization amount, and particularly when it is intended to analyze a gene by the hybridization method, the sensitivity is also good, and the glass plate is quantitative and reproducible. It has excellent properties and can be used repeatedly.

Further, as a method for analyzing nucleic acid using the above glass plate, there is an application to nucleotide sequence analysis. For example, determination of DNA nucleotide sequence using the hybridization method and diagnosis of infectious diseases and genetic diseases can be performed. These analysis methods can be applied to the mapping of giant genomic DNA, gene expression monitoring, and the like.

Specifically, as a method for diagnosing an infectious disease, for example, DNA is extracted from the blood of a subject and the DNA is extracted.
On the other hand, there is a method of detecting the presence of a pathogen by preparing a DNA probe from the sequences unique to various pathogens by the method of the present invention and performing a hybridization reaction.

As a method for diagnosing a genetic disease, an oligonucleotide is prepared on the basis of a sequence specific to a gene causing the genetic disease, and the oligonucleotide is hybridized with a chromosomal DNA obtained from a subject, and There is a method of detecting the presence or absence of mutation.

Further, although the giant gnome DNA mamping is an indispensable technique for a genomic DNA analysis project and the like, each of the clones can be cloned by hybridizing a large number of DNA probes prepared by this method with the DNA in the genomic bank. The arrangement on the genome can be efficiently determined.

The nucleic acid-immobilizing substrate of the present invention is not limited to the above embodiment.

FIG. 4 is a cross-sectional view of an essential part showing the second embodiment of the nucleic acid immobilizing substrate. In the second embodiment, a photoactive substance (for example, TiO ₂ ) and a water-repellent group are used. A mixed layer 6 in which the contained organic compound such as aminosilane is mixed is formed, and the mixed layer 7 is divided into a first region 7b having a high wettability and a second region 7a having a low wettability. The nucleic acid-immobilizing compound film 4 is formed on the surface of the region 7b.

That is, by exposing the hybrid layer 7 to the photomask 5 similar to that of the first embodiment and irradiating it with light, the wettability of the exposed portion is increased and the wettability of the first region 7b is increased. The second region 7a having a low wettability is formed, and as a result, the first region having a high wettability is formed as in the first embodiment.
The nucleic acid-immobilizing compound film can be easily formed on the surface of the region 7b.

[0057]

EXAMPLES Next, examples of the present invention will be specifically described.

First, the inventor of the present invention has made Ti as a photoactive substance
An O ₂ sol was prepared. That is, titanium n-butoxide was used as the organic titanium compound, 0.1 mol of this titanium n-butoxide was diluted with 16 mol of ethyl alcohol, and 0.1 ml of this diluted solution was added to ethyl acetoacetate.
Molar was added to stabilize. And 0.01 nitric acid
0.2 mol of dilute nitric acid dissolved in water at a concentration of mol / l was added to the diluted solution and stirred to prepare a TiO ₂ sol.

Next, a non-alkali glass substrate made of an aluminoborosilicate glass composition having a size of 25 mm × 76 mm × 1.1 mm is dipped in the TiO ₂ sol bath, and the pulling rate is 1.1 mm / The glass substrate is subjected to a dip coating treatment by pulling up the glass substrate for sec, and then at a temperature of 500 ° C. for 30 seconds.
A heat treatment was performed for minutes to form a photocatalyst layer (TiO ₂ film) made of anatase type titanium oxide with a film thickness of about 20 mm on the surface of the glass substrate.

Next, heptadecafluorodecyltrimethoxysilane (hereinafter referred to as "F") containing a fluoroalkyl group which is a water-repellent group as an organic compound which is decomposed by photocatalysis.
AS ”) and 0.01 mol of the FAS was diluted with 2.0 mol of isopropyl alcohol, and then 0.06
0.02 mol of dilute nitric acid of weight% was added and stirred in a flask to prepare FAS sol.

Then, the glass substrate on which the photocatalyst layer (titanium oxide film) was formed and the FAS sol were stored in a closed electric furnace and subjected to heat treatment at a temperature of 240 ° C. for 30 minutes, whereby the film thickness was several. nm thin film (FAS film) was laminated on the photocatalyst layer.

Next, the inventor has found that the center-to-center distance is 175 μm.
A photomask was prepared in which 2,000 m-shaped holes each having a diameter of 150 μm were provided in a grid pattern. Then, while covering the thin film (FAS film) with the photomask, an ultra-high pressure mercury lamp (manufactured by Ushio Inc .; “UIS-25102” (250 W, light wavelength 250 nm to 450 nm)) was used, and a wavelength of 220
~ 310 nm, illuminance 23.0 mW / cm ² , and wavelength 3
Light having a wavelength of 10 nm to 390 nm and an illuminance of 73.0 mW / cm ² was irradiated from above for 5 minutes.

As a result, the wettability does not change in the non-exposed area that is shielded by the photomask and is not irradiated with light. However, in the exposed area that is irradiated with light, the fluoroalkyl chain of FAS is decomposed by the photocatalytic action. When evaporated, FAS is converted to hydroxysiloxane or hydroxypolysiloxane, and the wettability is increased. That is, the light irradiation through the photomask forms an array pattern in which the first regions having high wettability and the second regions having low wettability are regularly arrayed on the glass substrate.

Water is dropped on the exposed and non-exposed portions of the thin film (FAS film), and the contact angle θ (8 in the figure indicates a water droplet) is measured by a contact angle meter (see FIG. 5). When measured by Wakiwa Interface Science Co., Ltd .; CA-DT), the contact angle θ of the non-exposed portion was as large as 108 °, whereas the contact angle θ of the exposed portion was
Was as small as 5 °, and therefore, it was confirmed that the wettability of the surface of the exposed area was higher than that of the unexposed area.

Next, the present inventor laminated a nucleic acid fixing compound on the first region of the patterned glass substrate.

That is, each patterned glass substrate was immersed in 350 ml of a poly-L-lysine solution for 1 hour, and then the glass substrate was centrifuged on a microtiter plate carrier at a rotation speed of 8.33 s ^-1 (500 rpm). After removing the liquid on the surface and further air-drying the glass substrate, the temperature was kept at 40 ° C. for 5 minutes and then stored at room temperature, whereby the nucleic acid fixing compound film was laminated on the first region to fix the nucleic acid. A substrate was obtained.

Next, DNA as nucleic acid was immobilized on the surface of the thus obtained substrate for nucleic acid immobilization to prepare a glass plate.

Specifically, first, the plasmid pGEM5Zf (+) (manufactured by US Promega Corporation) was used as DNA,
Labeling was performed with a fluorescent label (fluorescein) using LabelIT (Takara Shuzo).

Next, the thus labeled DNA was prepared to a concentration of 0.5 mg / ml, and 1 μl of the thus prepared DNA was applied to the nucleic acid-immobilizing substrate (poly-L-lysine-coated glass substrate). Dropped.

Then, D was dropped on the nucleic acid-immobilized substrate.
After air-drying NA, it was kept warm in a humid box at a temperature of 45 ° C. for 1 minute, and then this glass plate was subjected to an instantaneous drying treatment for 5 seconds on a hot plate having a temperature of 100 ° C., and then UV crosslinker (Funakoshi Co., Ltd. ) Was used to irradiate 60 mJ of ultraviolet light (wavelength 254 nm).

Next, the present inventor prepared an SMP solution,
The nucleic acid-immobilized substrate was immersed in the SMP solution for 10 minutes. That is, 5 g of succinic anhydride was dissolved in 315 ml of 1-methyl-2-pyrrolidone, and 35 ml of 0.2 M sodium borate solution (pH 8.0) was added to add SMP.
A solution was prepared, and the nucleic acid-immobilized substrate was immersed in the SMP solution thus prepared for 10 minutes.

Then, after washing with hot water of 95 ° C. for 2 minutes,
It was washed with 95% by weight of ethyl alcohol for 1 minute, and the substrate for nucleic acid immobilization was further centrifuged on a microtiter plate carrier at 8.3 s ^-1 (500 rpm) to remove the liquid on the glass surface.

On the other hand, the present inventor produced a substrate in which poly-L-lysine was directly laminated on a glass substrate, and DNA was formed on the substrate.
1 μl was dropped and the same treatment as described above was performed to prepare a glass plate of a comparative example.

In this way, the present inventor produced 30 glass plates for each of the Examples and Comparative Examples, and then quantified the fluorescence amount of DNA on the glass plates to evaluate the DNA immobilization state. . The amount of fluorescence was detected using a Fluorolmager (Molecular Dynamic Inc., USA).

The immobilization state of DNA in a desired region is evaluated by
The fluorescence intensity ratio between the dropped portion of the comparative example DNA and the peripheral portion thereof was set to "1", and evaluation was performed by relative comparison with the DNA of the example. As a result, it was confirmed that in Example, DNA was immobilized only in the desired region at a fluorescence intensity ratio about 10 times that in Comparative Example.

[0076]

As described above in detail, in the nucleic acid-immobilizing substrate according to the present invention, the array pattern in which the first region having high wettability and the second region having low wettability are regularly arrayed is formed by the glass. Since it is formed on the surface of the substrate, the nucleic acid is stably immobilized in the first region having high wettability while avoiding the nucleic acid from adhering to the second region having low wettability. It is possible to stably immobilize the nucleic acid in the desired region of

In the nucleic acid immobilizing substrate of the present invention, a nucleic acid immobilizing compound film containing a functional group of at least one of an amino group and a carboxyl group is formed on the surface of the first region. Therefore, the nucleic acid can be stably immobilized only in the first region easily.

Further, in the nucleic acid immobilizing substrate of the present invention, a photocatalytic layer made of a photoactive substance is formed on the surface of the glass substrate, and a thin film made of an organic compound which is decomposed by a photocatalytic action is laminated on the photocatalytic layer. The first region and the second region are patterned and formed on the thin film, or a mixed film in which a photoactive substance and an organic compound which is decomposed by photocatalysis is mixed is formed on the surface of the glass substrate. Since the first region and the second region are formed on the mixed film by patterning, the nucleic acid can be stably immobilized only on the first region having high wettability. it can.

In addition, the method for producing a substrate for immobilizing nucleic acid according to the present invention is a thin film made of an organic compound, which has a photocatalytic layer having photoactivity formed on the surface of a glass substrate and is decomposed by photocatalytic action to change wettability. And irradiating the thin film with light through a photomask having a predetermined pattern to form a pattern in which a first region having large wettability and a second region having small wettability are regularly arranged, Alternatively, after forming a mixed film containing a photocatalytic substance having photoactivity and an organic compound which is decomposed by a photocatalytic action and changes wettability on the surface of a glass substrate, the mixed film is exposed to light through the photomask having a predetermined pattern. Irradiation is performed to form a pattern in which the first region having high wettability and the second region having low wettability are regularly arranged. Therefore, the exposed thin film portion is separated by the photocatalytic action. Then, the wettability becomes large, and the nucleic acid immobilizing substrate in which the first area having the large wettability and the second area having the small wettability are arranged regularly is manufactured. be able to.

By using a fluoroalkyltrialkoxysilane, a hydrolyzate thereof, or a polycondensate thereof as the organic compound, the fluoroalkyl group which is a water-repellent group is decomposed by a photocatalytic action and evaporated,
This makes it possible to easily increase the wettability of the exposed portion.

By using titanium oxide as the photoactive substance, a desired photocatalytic action can be easily obtained.

Further, in the glass plate according to the present invention, the nucleic acid is immobilized on the first region formed on the nucleic acid immobilizing substrate, so that the nucleic acid is stably immobilized only on the first region. This makes it possible to easily obtain a nucleic acid array or nucleic acid chip having a desired regular array pattern.

Since the nucleic acid analysis method according to the present invention analyzes the nucleic acid immobilized on the nucleic acid immobilizing substrate of the glass plate, the nucleic acid sample regularly arrayed on the glass plate is analyzed. By doing so, it becomes possible to efficiently determine the nucleotide sequence and analyze various genes such as infectious diseases and genetic diseases.

That is, according to the present invention, nucleic acids such as DNA can be efficiently and stably immobilized only in a desired region on a glass substrate, and even when a gene is analyzed by a hybridization method, the nucleic acid can be immobilized. Since it is performed only in a desired area, it can be used for detection of a small amount of substance with high sensitivity, and because it is stably immobilized, it has excellent reproducibility and quantitativeness.

[Brief description of drawings]

FIG. 1 is a sectional view of essential parts showing an embodiment (first embodiment) of a nucleic acid-immobilizing substrate according to the present invention.

FIG. 2 is a manufacturing process diagram showing an embodiment of a method for manufacturing a nucleic acid-immobilizing substrate according to the present invention.

FIG. 3 is a cross-sectional view of an essential part of a glass plate, which schematically shows a state in which nucleic acids are immobilized in a desired region of the nucleic acid immobilizing substrate.

FIG. 4 is a cross-sectional view of essential parts showing a second embodiment of a nucleic acid-immobilizing substrate according to the present invention.

FIG. 5 is an explanatory diagram for explaining a method of measuring a contact angle.

[Explanation of symbols]

1 glass substrate 2 Photocatalyst layer 3 thin film 3a Second area 3b First area 4 Nucleic acid immobilization compounds 5 photo mask 6 Nucleic acid 7 Mixed film formation

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 37/00 102 C12N 15/00 F

Claims (10)

[Claims] 1. An array pattern in which a first region having high wettability and a second region having low wettability are regularly arrayed,
A nucleic acid-immobilizing substrate, which is formed on the surface of a glass substrate. 2. The nucleic acid-immobilizing compound film containing a functional group of at least one of an amino group and a carboxyl group is formed on the surface of the first region. The nucleic acid-immobilized substrate described. 3. A photocatalyst layer made of a photoactive substance is formed on the surface of the glass substrate, and a thin film made of an organic compound which is decomposed by a photocatalytic action is laminated on the photocatalyst layer to form the first region and the first region. 2. The region 2 is formed by patterning on the thin film.
Alternatively, the nucleic acid-immobilizing substrate according to claim 2. 4. A mixed film in which a photoactive substance and an organic compound decomposed by a photocatalytic action are mixed is formed on the surface of the glass substrate, and the first region and the second region are formed on the mixed film. The nucleic acid-immobilizing substrate according to claim 1 or 2, which is formed by patterning. 5. A photocatalytic layer having photoactivity is formed on the surface of a glass substrate, and then a thin film made of an organic compound which is decomposed by a photocatalytic action and changes in wettability is laminated, and the photocatalytic layer is formed through a photomask having a predetermined pattern. A method for producing a nucleic acid immobilizing substrate, which comprises irradiating a thin film with light to form a pattern in which first regions having high wettability and second regions having low wettability are regularly arranged. 6. A mixed film containing a photocatalytic substance having photoactivity and an organic compound which is decomposed by a photocatalytic action and changes in wettability is formed on the surface of a glass substrate, and then the film is formed through a photomask having a predetermined pattern. Light irradiation for mixed film formation,
A method for producing a nucleic acid immobilizing substrate, which comprises forming a pattern in which first regions having high wettability and second regions having low wettability are regularly arranged. 7. The method for producing a nucleic acid immobilizing substrate according to claim 5, wherein the organic compound is fluoroalkyltrialkoxysilane, a hydrolyzate thereof, or a polycondensate thereof. 8. The method for producing a substrate for nucleic acid immobilization according to claim 5, wherein the photoactive substance is titanium oxide. 9. A glass plate, wherein nucleic acid is immobilized on the first region formed on the nucleic acid immobilizing substrate according to any one of claims 1 to 4. 10. A method for analyzing nucleic acid, which comprises analyzing the nucleic acid immobilized on the nucleic acid-immobilizing substrate of the glass plate according to claim 9.