JP2003254972A - Probe medium and method for fixing probe on substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently and stably fix probes on a substrate. <P>SOLUTION: A probe specifically joinable to a target substance and a probe medium including a substance for fixing the probe on a substrate, the probe specifically joinable to the target substance and a probe medium including joint substances of two or more kinds including a substance A joinable to the probe and substance B joinable to the substrate, and the probe specifically joinable to the target substance and a probe medium including a water-soluble polymer material, and provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a probe medium for fixing a probe on a substrate and a method for fixing the probe medium. The present invention also relates to a detection method for detecting a target substance using a probe-immobilized substrate obtained by immobilizing a probe medium on a substrate.

[0002]

2. Description of the Related Art Various methods are known for fixing a probe on a substrate. Specifically, there are a method of fixing the probe on the base material by synthesizing the probe on the base material, and a method of applying the probe prepared in advance on the base material with a pin or a stamp. Specifically, by removing the protecting group from a selected region of the substrate with an activator and attaching a monomer having a removable protecting group to the region repeatedly, various sequences are arranged on the substrate. There is known a method of synthesizing a polymer having a polymer (for example, Patent Document 1).
reference). In addition, the material for immobilization consisting of the base material and the polymer compound having a carbodiimide group carried on the base material and the biologically active substance reactive with the carbodiimide group are contacted for immobilization. A method is known (for example, refer to Patent Document 2). Similarly, a method for detecting a biologically active substance by immobilizing it on a compound having a carbodiimide via a carbodiimide group is known (see, for example, Patent Document 3). Furthermore, DN having a thiol group at the end
By contacting in the liquid phase, the A fragment and a solid phase carrier having a chain molecule having a reactive substituent capable of reacting with the thiol group to form a covalent bond are immobilized on the surface at one end, There is known a method for immobilizing a DNA fragment on the surface of a solid phase carrier by forming a covalent bond between the DNA fragment and a chain molecule (see, for example, Patent Document 4).

Many methods are also known as methods for detecting biological components. A method has been described for testing substances such as biological components such as RNA, cDNA, oligonucleotides, etc., with high sensitivity and high accuracy, while eliminating the influence of background noise (for example, Patent Document 5).
reference). Further, there is known a method of fixing a reactive substance on a substrate by ejecting a solution containing the reactive substance from a nozzle to a desired position on the substrate to be reacted (for example, see Patent Document 6). The substrate is made of glass or silicon, the surface of which has been surface-treated to give affinity, and the reactive substance is DNA fragment, cDNA.
It is at least one selected from the group consisting of A, RNA, enzyme, antigen, antibody, epitope, protein, polynucleotide, and peptide.

It is known to use a water-soluble polymeric material for blocking treatment when fixing a probe on a substrate. Specifically, it is a method of immobilizing a sample nucleic acid on a membrane and then immersing it in a solution containing PVA or / and PVP for blocking treatment (see, for example, Patent Document 7). other,
Regarding the treatment after fixing the nucleic acid on the membrane, it is known to perform a blocking treatment using skim milk or the like. Specifically, after fixing the sample nucleic acid to the membrane, the membrane is subjected to a blocking treatment with a solution obtained by reacting skim milk with N-succinimidyl-3- (2-pyridylthio) propionate (for example, Patent Document 8). reference).

Further, in a probe-immobilized substrate in which the probe is immobilized on the substrate, after the probe is applied on the substrate, D
It is known to prevent drying of NA probes. Specifically, a high boiling point solvent is added to an aqueous solution in which DNA fragments are dissolved or dispersed (see, for example, Patent Document 9).

Furthermore, with respect to coating medical devices with polymeric materials, it is known to coat medical devices with polymers and drugs. Specifically, a composition for coating a medical device including a biocompatible biodegradable polymer and a drug is known (see, for example, Patent Document 10).

[0007]

[Patent Document 1] US Pat. No. 5,138,545

[Patent Document 2] JP-A-8-23975

[Patent Document 3] Japanese Patent Laid-Open No. 8-334509

[Patent Document 4] Japanese Patent Laid-Open No. 2001-178442

[Patent Document 5] Japanese Patent Laid-Open No. 2001-116699

[Patent Document 6] Japanese Patent Laid-Open No. 2001-116750

[Patent Document 7] Japanese Patent No. 2794728

[Patent Document 8] Japanese Patent Publication No. 06-034756

[Patent Document 9] Japanese Patent Laid-Open No. 2001-178459

[Patent Document 10] Special Table 2000-512519

[0008]

However, as a probe medium containing a probe that can specifically bind to a target substance, which has been conventionally used, a probe medium containing a probe and a water-soluble polymer material in one probe medium is used. There was no. It has been known that a water-soluble polymer material is used in the blocking treatment which is one step of the detection method of detecting a target substance using a probe-immobilized substrate, but the probe medium contains the water-soluble polymer material. There was no such thing as mixing the probe and the water-soluble polymer material when fixing the probe on the substrate. Further, as a probe-immobilized substrate in which a probe capable of specifically binding to a target substance, which has been conventionally used, is immobilized on a substrate, a DNA fragment which is one of the probes is immobilized on the substrate and then DNA It was known to prevent the pieces from drying out. Therefore, it is necessary to add a high boiling point solvent to an aqueous solution in which DNA fragments are dissolved or dispersed.

[0009]

The above problems have been solved by the present invention described below. (1) A probe medium containing a probe capable of specifically binding to a target substance, a water-soluble polymer material, and a high boiling point solvent. The water-soluble polymer material may be polyvinyl alcohol. The high boiling point solvent may be a glycol-based high boiling point organic solvent. The probe may be a DNA probe. With this probe medium, it was possible to prepare a probe, a water-soluble polymer material and a high boiling point solvent contained in one medium. (2) A probe fixing method in which the probe medium is fixed by applying it on a substrate by a spotting method.
A DNA array in which the probe of the probe-immobilized substrate produced by the probe immobilization method is a DNA probe. A detection method for detecting a target substance using the probe-immobilized substrate obtained by the present probe immobilization method. (3) A probe medium in which the probe, the water-soluble polymer material, and the high-boiling-point solvent are each placed in a container and mixed when fixed on a substrate.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In a probe medium and a method for immobilizing a probe on a substrate according to an embodiment of the present invention, a probe, a water-soluble polymer material and a medium containing a high boiling point solvent are used.

As the probe, a single-stranded nucleic acid probe,
A single-stranded DNA probe, a single-stranded RNA probe, a single-stranded PNA probe and the like are included. The amount of the probe contained in the medium is, for example, 2 mer to 50 in the medium in consideration of the stability of the nucleic acid probe as the probe medium.
0 mer, particularly 2 mer to 80 mer nucleic acid probe, 0.05 to 500 μmol / l, especially 0.5 to 5
It is preferable to adjust the concentration to 0 μmol / l. These probes may have a functional group. For example, as the functional group, an amino (NH2) group,
Examples thereof include functional groups such as a carbonyl (COOH) group, a mercapto (SH) group, and a hydroxyl (OH) group.

As the water-soluble polymer material, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PV
P), paogen, carboxymethyl cellulose (CM
C), hydroxyethyl cellulose (HEC), dextran, pullulan and the like. Among these, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PV
P) is a general-purpose material and is preferable because it is stable with respect to the probe. In the case of polyvinyl alcohol (PVA), which is a general-purpose material, the degree of polymerization
It is preferable to adjust the degree of saponification. By adjusting the degree of polymerization and saponification, it is possible to adjust the solubility of polyvinyl alcohol in the probe medium, the viscosity of the probe medium, the spot strength after spotting the probe medium on the substrate, the hygroscopicity, etc. Becomes Specifically, it is preferable to adjust the degree of polymerization and the degree of saponification so that the probe stability becomes appropriate, if there is no problem in providing the substrate as a probe medium. Furthermore, the spot shape characteristics and hygroscopicity of the probe-immobilized substrate formed by applying the probe medium onto the substrate may be adjusted to a suitable state. But,
When the degree of polymerization of polyvinyl alcohol is high,
Not only does the viscosity of the probe medium improve, but
Since the solubility of polyvinyl alcohol in the probe medium decreases, it becomes difficult to apply it to the substrate as a stable probe medium. When the degree of saponification of polyvinyl alcohol is high, not only the solubility of polyvinyl alcohol in the probe medium is lowered, but also the viscosity of the probe medium is improved, which makes it difficult to apply the polyvinyl alcohol onto the substrate. As a method of avoiding this, there is a method of lowering the concentration of polyvinyl alcohol, but probe stability, spot shape characteristics, hygroscopicity, etc. are reduced due to the concentration decrease, so it is necessary to select appropriate polyvinyl alcohol and adjust the concentration. . It is preferable to adjust the concentration of polyvinyl alcohol in the probe medium to be 0.001 to 1 wt%. Especially 0.01-0.2 wt%
It is preferable to adjust so that When the polyvinyl alcohol concentration in the probe medium is within the above range, it is preferable to lower the degree of polymerization of polyvinyl alcohol and increase the degree of saponification. The polymerization degree and saponification degree of polyvinyl alcohol are preferably, for example, 200 to 1700 and 87 to 99 mol%, respectively.

Such polyvinyl alcohol may be dissolved in advance and mixed with the probe medium. The medium to be dissolved may be an organic solvent such as ethanol or dimethylsulfoxide (DMSO), but water and ethanol are preferable when solubility is taken into consideration. As a method for dissolving, insoluble matter can be dissolved quickly by mixing polyvinyl alcohol in water and then heating. It is preferable to filter the dissolved polyvinyl alcohol so that there is no insoluble matter, and then mix with the probe medium. Regarding the solution in which polyvinyl alcohol is dissolved, if the stability of the probe when mixed with the probe is taken into consideration, pH 6.0 to pH
It is 9.0, and it is particularly preferable to adjust the pH to 7.0 to 8.0.

The probe medium containing the probe and the water-soluble polymer material preferably contains water,
In addition to water, organic solvents such as ethylene glycol, thiodiglycol, glycerin and isopropyl alcohol may be included in the medium.

As the high boiling point solvent, ethylene glycol, propylene glycol, triethylene glycol,
Examples include diethylene glycol, dipropylene glycol and thiodiglycol. Among these, thiodiglycol and dipropylene glycol are versatile materials,
It is preferable because it is also suitable as a spotting medium. Since the glycol-based high boiling point organic solvent is a highly viscous liquid, it is difficult to weigh the solvent during mixing. Therefore, it may be mixed in advance with a low-viscosity liquid such as isopropyl alcohol. Examples of the low-viscosity liquid mixed at this time include alcohol solvents other than isopropyl alcohol, water, and the like. For the addition of high boiling point solvent,
It is preferable to adjust the spot shape characteristics, hygroscopicity, dryability, and dry state when applied as a probe medium onto a substrate so as to be suitable.

As a concrete medium, water is 70 to 95 w.
t%, thiodiglycol 0.1-3%, isopropyl alcohol 5-30 wt%, polyvinyl alcohol 0.0
Those containing 0 to 1 wt% are included. As a more suitable medium, water is 80 to 90 wt%, thiodiglycol is 1 to 2.5 wt%, and isopropyl alcohol is 10 to 20.
wt%, polyvinyl alcohol 0.01-0.2 wt%
Is.

Further, the probe medium may contain a probe fixing substance in order to efficiently fix the probe on the substrate. As a probe fixing substance, a primer, a coupling agent, a sealant, a surface treatment agent,
It includes surface modifiers, binding agents for coupling agents and cross-linking agents, binding agents for coupling agents and divalent reagents, and the like. The amount of the substance contained in the medium is, for example, 0.05 to 5 in the medium in consideration of the immobilization property of the probe on the substrate.
0000 μmol / l, especially 10-500 μmol / l
It is preferable to adjust the concentration to be l. These substances may have a functional group that binds to the probe or the probe functional group, and as the functional group, an epoxy (CHOCH2) group, an amino (NH2) group, a mercapto (SH) group, a maleimide group, chloropropyl group. (C3
H6Cl) group, isocyanate (NCO) group and the like are included. In particular, the probe functional group is an amino (NH2) group,
A carbonyl (COOH) group, a mercapto (SH) group, a hydroxyl group (OH) group and the like are preferable, and as the functional group capable of binding to the probe, an epoxy group, a maleimido group, a chloropropyl group is considered in consideration of the binding with the probe functional group. Groups are preferred. The substance that fixes these probes on the substrate may have a functional group for fixing the probe on the substrate, and the functional group may be alkoxysilyl (SiO 2).
R) group, silanol (SiOH) group, alkoxy (O
R) groups and the like are included. In particular, an alkoxysilyl group and a silanol group are preferable because they are easily fixed on the substrate. The silanol group may be formed by hydrolysis of an alkoxysilyl group. The probe and the substance that fixes the probe on the substrate may be bound by reacting in a medium, and the reaction is preferably a covalent bond due to a chemical reaction between functional groups.

As a method of mixing the water-soluble polymer material with the probe medium, a solution having a predetermined concentration in which the water-soluble polymer material is completely dissolved is prepared in advance. A method in which the prepared water-soluble polymer solution is weighed so as to have a predetermined concentration and mixed is suitable. As the water-soluble polymer material solution, specifically, polyvinyl alcohol powder is weighed and added to pure water in an amount of 0.
Mix so as to have a concentration of 5 to 5 wt%. After the polyvinyl alcohol is dissolved while heating the mixed solution, it is filtered for the purpose of removing insoluble matters and foreign substances. In this way, a water-soluble polymer aqueous solution is prepared. This water-soluble polymer aqueous solution is preferably mixed in the probe medium so that the concentration in the probe medium is 0.01 to 0.2 wt%.

The procedure for mixing the water-soluble polymer material into the probe medium is not particularly limited, but it is preferably the last step of mixing the probe with another medium.
As a method of mixing the high boiling point solvent with the probe medium, a method of preliminarily weighing so as to have a predetermined concentration and then mixing is preferable.

The procedure for mixing the high boiling point solvent into the probe medium is not particularly limited, but it is preferable that the probe medium is mixed with the water-soluble polymer at the end of mixing the probe with another medium.

The probe medium thus obtained is
It is suitable as a probe medium for detecting a target substance.

A probe-immobilized substrate can be prepared by spotting the obtained probe medium on the substrate. The substrate for immobilizing the probe is not particularly limited, but a glass or quartz substrate material is preferable in view of detection of the target substance and versatility as a material. As a concrete material, 1 inch × 3 inch
A ch-sized slide glass substrate is preferable, and in consideration of the fixing characteristics of probe fixing, it is preferable to use a non-alkali material slide glass substrate or a quartz glass material in which the glass material does not contain an alkaline component or the like. . Further, the shape of the substrate or the like is not limited depending on the method of detecting the target substance, but the substrate state is preferable in terms of versatility of the detection method and apparatus. Furthermore, it is desired that the substrate material has a high surface smoothness.

Further, the base material for fixing the probe is preferably a clean surface in order to uniformly and surely fix the probe on the base material. It is desirable to secure a sufficient clean surface by cleaning the base material in advance before fixing the probe. Many methods such as water cleaning, chemical cleaning, plasma cleaning, and UV ozone cleaning are known as methods for cleaning the base material, but as a simple and uniform cleaning method, a chemical solution is used. The washing method by is suitable. For example, there is a method in which the surface of the base material is thoroughly washed with an aqueous sodium hydroxide solution having a predetermined concentration to remove the dirt attached to the base material. Specifically, a 1 mol / l sodium hydroxide aqueous solution heated to about 50 ° C. is prepared, and the surface of the substrate is wiped or brushed while showering the aqueous solution on the substrate. Make sure to remove the dirt that has adhered. After removing the dirt,
Wash off excess sodium hydroxide thoroughly with water. Finally, water removal may be performed by N2 blowing or the like. In this way, it is possible to obtain a base material on which the probe medium can be fixed uniformly and surely.

If the probe medium does not contain a probe immobilization substance, it is preferable that the probe immobilization treatment is performed on the substrate. Specifically, it means that the substrate which has been sufficiently washed as described above is treated with the silane coupling agent. For example, the washed silane coupling agent is immersed in a solution in which a silane coupling agent is sufficiently stirred in a mixed solvent of water and ethyl alcohol, and then the washed substrate is immersed in the solution to remove excess silane coupling agent and dried. It is a base material.
Further, a solution prepared by adding a silane coupling agent to ethyl alcohol is sufficiently stirred to prepare a silane coupling agent solution, which is coated on a substrate washed by a spin coating method.

There are several known methods for spotting the probe medium on the substrate. Specific methods include pin method, inkjet method, pin &
The ring method is known. Among these, the inkjet method is a preferable spotting method because it has high density and can perform accurate spotting.

In the spotting method by the ink jet method, the components contained in the probe medium substantially affect the probe and the water-soluble polymer material when ejected from the ink jet head as the probe medium as described above. It is not particularly limited as long as it does not exist and satisfies the medium composition that can be normally ejected onto the substrate using the inkjet head. For example, when the inkjet head is a bubble jet (registered trademark) head having a mechanism for applying thermal energy to a medium and ejecting the medium, a liquid containing glycerin, thiodiglycol, and isopropyl alcohol is preferable as a component contained in the probe medium. Is. More specifically, glycerin 5 to 10 wt
%, And a liquid containing 5 to 10 wt% of thiodiglycol is suitably used as the probe medium. Further, when the inkjet head is a piezo jet head which ejects a medium by using a piezoelectric element, a liquid containing ethylene glycol and isopropyl alcohol is preferable as a component contained in the probe medium. More specifically, a liquid containing 5 to 10 wt% of ethylene glycol and 0.5 to 2 wt% of isopropyl alcohol is preferably used as the probe medium.

When the probe medium thus obtained is ejected from the ink jet head and adhered to the substrate, the spot shape is circular and the ejected range does not spread. Even when the probe medium is spotted at a high density, the connection with the adjacent spots can be effectively suppressed. Further, even when the probe medium is dried and the spot is dried, the probe is stored without any problem and the target substance can be detected. Furthermore, it is possible to easily observe the spot state even after drying, and it is possible to confirm whether or not the spot was formed without any problem in spotting.
The characteristics of the probe medium of the present invention are not limited to the above.

The probe and the base material are preferably bound by reacting between the probe and the base material. By reacting, the probe is firmly fixed on the base material. As a result, the probe is immobilized on the substrate, and the spot of the probe can be formed at a predetermined position. The water-soluble polymer material has no problem in fixing the probe on the substrate, and by adding the water-soluble polymer material to the probe medium, it has the effect of delaying spot drying when spotting, There is an effect that the reaction between the probe and the substrate is sufficiently performed.

For example, a probe medium containing a probe having a base length of 20 mer was adjusted to have a probe concentration of 8 μmol / l. As the probe medium, a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc., but modified to print on a flat plate) was used, and the solid phase and the nozzle of the bubble jet (registered trademark) head were used. When the distance is set to about 1.2 to 1.5 mm and the ink is discharged from the nozzle (discharge amount is about 4 picoliters), a spot having a diameter of about 50 to 80 μm can be formed on the solid phase. Also, no spots (hereinafter referred to as "satellite spots") derived from the droplets when the liquid lands on the solid-phase surface were not observed at all by visual observation using a loupe.

A Fourier-transform infrared spectrophotometer for ATR microscopic measurement (ATR microscopic FT-I) is prepared by using the above-mentioned probe-immobilized substrate (on which a probe medium containing a probe-immobilized substance is immobilized).
The analysis was carried out according to R). At sites other than the spots, Si—O bond peaks due to the substrate could be detected, but other bond peaks were hardly observed. On the other hand, in addition to being able to detect the broad peak of the base material at the spot forming site, the peak of -CH2-bond of the probe fixing substance and the water-soluble polymer material contained in the probe medium was detected. We were able to.

In addition, the probe-immobilized substrate is subjected to a temperature desorption method (TDS: Thermal Desorption Spec).
The analysis was performed by trometry). 7m including the part where the spot is formed when performing the analysis
An m □ substrate piece and a 7 mm □ substrate piece on the spot peripheral portion where no spot was formed were cut and extracted for comparison. The temperature was 25 to 500 ° C., and the temperature rising rate was 5 ° C. per minute.
As a result, when the desorption component masses of both substrate pieces were compared,
At the spot formation site, a large amount of mass was detected at a temperature of 150 ° C. or higher due to a probe such as CH4 and CH3OH and a substance that fixes the probe on the substrate. From these facts, it was confirmed that the probe medium was spotted only on the spot portion, and the probe medium and the substance contained in the medium were not applied to other than the spot portion.

A Fourier-transform infrared spectrophotometer for ATR microscopic measurement (ATR microscopic FT-) was prepared by using the above-mentioned probe-immobilized substrate (immobilized probe medium containing no probe-immobilized substance).
The analysis was carried out by IR). At sites other than the spots, Si—O bond peaks due to the substrate could be detected, but other bond peaks were hardly observed. On the other hand, at the spot formation site, in addition to being able to detect the broad peak of the base material, the peak of the -CH2-bond of the water-soluble polymer material contained in the probe medium could be detected. It was

Further, the probe fixing base material is subjected to a temperature desorption method (TDS: Thermal Desorption Spec).
The analysis was performed by trometry). 7m including the part where the spot is formed when performing the analysis
An m □ substrate piece and a 7 mm □ substrate piece on the spot peripheral portion where no spot was formed were cut and extracted for comparison. The temperature was 25 to 500 ° C., and the temperature rising rate was 5 ° C. per minute.
As a result, when the desorption component masses of both substrate pieces were compared,
At the spot formation site, a large amount of mass was detected at a temperature of 150 ° C. or higher due to a probe such as CH4 and CH3OH and a substance that fixes the probe on the substrate. From these facts, it was confirmed that the probe medium was spotted only on the spot portion, and the probe medium and the substance contained in the medium were not applied to other than the spot portion.

Here, using this probe-immobilized substrate,
For example, the detection accuracy (S /
For the purpose of improving the (N ratio), after fixing the probe on the solid phase surface, blocking is performed so that the probe non-binding portion on the substrate does not bind to the target substance contained in the sample. Is also good. Blocking is for example
The base material is placed in a 2% bovine serum albumin aqueous solution, for example, 2
It is done by soaking for about an hour. However, the bovine serum albumin aqueous solution is preferable in terms of the effect of preventing the adsorption of the labeling substance on the substrate other than the probe-immobilized site. It should be noted that this blocking step may be carried out as necessary. For example, the sample is supplied to the probe-immobilized substrate in a limited manner for each spot, and the sample is substantially adhered to the site other than the spot. If not, you don't have to do it. The adhesion of the sample to the site other than the spot depends on the material used as the base material. In particular, when the substrate is glass or quartz and the probe medium contains a probe immobilization substance, the blocking treatment may not be performed. However, when the probe immobilization substance is not contained in the probe medium and the probe immobilization treatment is performed on the substrate, it is preferable to perform the blocking treatment.

The probe-immobilized substrate thus produced may have a plurality of spots containing the same probe, for example, or may have a plurality of spots containing different kinds of probes, depending on the application. It may be configured like this. The type, quantity, and arrangement of probes can be changed as needed. And the probe-immobilized substrate on which the probes are arranged in high density by such a method is
Then, it is used for detection of the target substance, specification of the target substance base sequence, and the like. For example, when it is used to detect a single-stranded nucleic acid that is a target substance having a known base sequence that may be contained in a sample, it is complementary to the base sequence of the single-stranded nucleic acid of the target substance. A single-stranded nucleic acid having a unique base sequence is used as a probe, and a probe-immobilized substrate having a plurality of spots containing the probe arranged on a solid phase is prepared. Is supplied and placed under conditions such that the single-stranded nucleic acid of the target substance and the probe hybridize, and then the presence or absence of hybrid in each spot is detected by a known method such as fluorescence detection. Thereby, the presence or absence of the target substance in the sample can be detected.

Further, when it is used for specifying the base sequence of the single-stranded nucleic acid of the target substance contained in the sample, a plurality of candidates for the base sequence of the single-stranded nucleic acid of the target substance are set, A single-stranded nucleic acid having a base sequence complementary to the base sequence group is spotted on the substrate as a probe. Then, a sample is supplied to each spot and placed under conditions such that the single-stranded nucleic acid of the target substance and the probe hybridize, and then the presence or absence of a hybrid in each spot is determined by a known method such as fluorescence detection. Detect with. Thereby, the base sequence of the single-stranded nucleic acid of the target substance can be specified. Further, other uses of the probe-immobilized substrate according to the present invention include application to screening of a specific base sequence recognized by a DNA-binding protein and screening of a chemical substance having a property of binding to DNA.

The probe medium is not limited to the one containing the probe and the water-soluble polymer material as described above, but the probe and the water-soluble polymer material are housed in respective containers and fixed on the substrate. It may be mixed at that time.

As the probe, a single-stranded nucleic acid probe,
A single-stranded DNA probe, a single-stranded RNA probe, a single-stranded PNA probe and the like are included. The amount of the probe contained in the medium is, for example, 2 mer to 50 in the medium in consideration of the stability of the nucleic acid probe as the probe medium.
0 mer, particularly 2 mer to 80 mer nucleic acid probe, 0.05 to 500 μmol / l, especially 0.5 to 5
It is preferable to adjust the concentration to 0 μmol / l. These probes may have a functional group. For example, as the functional group, an amino (NH2) group,
Examples thereof include functional groups such as mercapto (SH) group and hydroxyl (OH) group.

When storing the probe in the container, the container is preferably a container that can be hermetically sealed so as not to mix other impurities.
It must be openable so that it can be easily mixed when fixed. The opening as a container is not particularly limited. The state of the probe housed in the container is not particularly limited, but it may be in the form of solution or powder. When the functional group is a mercapto group or the like, it is preferable to store it as powdered by a freeze-dry method or the like in consideration of the stability of the probe. The storage state is preferably frozen for ensuring the stability of the probe, but the storage state can be changed according to the storage period, probe type, probe functional group, and the like. As described above, the probe may be stored in the container and mixed when the probe is stored and fixed on the substrate.

As the water-soluble polymer material, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PV
P), paogen, carboxymethyl cellulose (CM
C), hydroxyethyl cellulose (HEC), dextran, pullulan and the like. Among these, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PV
P) is a general-purpose material and is preferable because it is stable with respect to the probe. In the case of polyvinyl alcohol (PVA), which is a general-purpose material, the degree of polymerization
It is preferable to adjust the degree of saponification. By adjusting the degree of polymerization and the degree of saponification, it becomes possible to adjust the solubility of polyvinyl alcohol in the probe medium and the viscosity of the probe medium. Specifically, if there is no problem in applying the probe medium on the substrate,
It is preferable to adjust the degree of polymerization and the degree of saponification so that the probe stability becomes appropriate. However, when the degree of polymerization of polyvinyl alcohol is high, and when the degree of saponification is high, not only the viscosity as a probe medium is improved, but also the solubility of polyvinyl alcohol in the probe medium is reduced, and therefore, as a probe medium. Dissolution becomes difficult when adjusting. As a method of avoiding this, there is a method of lowering the concentration of polyvinyl alcohol, but probe stability, spot shape characteristics, hygroscopicity, etc. are reduced due to the concentration decrease, so it is necessary to select appropriate polyvinyl alcohol and adjust the concentration. . The concentration in the probe medium as polyvinyl alcohol is 0.001-1 wt.
It is preferable to store in a container so that the ratio becomes%.

When the dissolution is difficult as described above, the water-soluble polymer material may be dissolved in advance and mixed with the probe medium. The medium to be dissolved may be an organic solvent such as ethanol or dimethylsulfoxide (DMSO), but in consideration of solubility,
Water and ethanol are preferred. As a method for dissolving, insoluble matter can be dissolved quickly by mixing polyvinyl alcohol in water and then heating. It is preferable to filter the dissolved polyvinyl alcohol so that there are no impurities or insolubles and then mix with the probe medium. Regarding the solution in which polyvinyl alcohol is dissolved, if the probe stability when mixed with the probe is considered, it is pH 6.0 to pH 9.0, and particularly pH
It is preferable to adjust the pH to 7.0 to 8.0.

When the water-soluble polymer material is stored in the container, the container should be hermetically sealed so that other impurities and the like will not be mixed in and that the amount will not be reduced due to volatilization of the substance. It is preferable that it is possible or a closed container, and it is preferable that it can be easily opened so that mixing is easy when fixing. As the storage state, it is preferable to store at room temperature in order to ensure the stability of the substance, but the storage state can be changed according to the storage period and the like. In particular, in the case of a water-soluble polymer material in solution, it is preferable that the temperature during storage does not rise. In addition, it is preferable to obtain probe stability by adjusting the pH according to the functional group of the substance that fixes the probe on the substrate.

As the high boiling point solvent, ethylene glycol, propylene glycol, triethylene glycol,
Examples include diethylene glycol, dipropylene glycol and thiodiglycol. Among these, thiodiglycol and dipropylene glycol are versatile materials,
It is preferable because it is also suitable as a spotting medium. The addition as a high boiling point solvent is preferably adjusted so that the spot shape characteristics, hygroscopicity, dryability, etc. when applied as a probe medium on a substrate are in a suitable state.

When storing the high boiling point solvent in a container,
It is preferable that the container can be hermetically sealed so that other impurities and the like are not mixed in, and further, that the amount is not reduced due to volatilization of the substance, or a hermetically sealed container is preferable. It is preferable that it can be easily opened so that mixing is simple when fixing. As the storage state, it is preferable to store at room temperature in order to ensure the stability of the substance, but the storage state can be changed according to the storage period and the like. Particularly, in the case of a high boiling point solvent, it is preferable that the temperature during storage does not rise.

When fixing the probe on the base material, a solution such as water may be added, if necessary, in mixing the probe and the substance for fixing the probe housed in these containers. Alternatively, a substance that is sufficiently mixed to form the probe medium, for example, a solvent such as water may be stored in a separate container and mixed when preparing the probe medium.

The present invention will be described in more detail with reference to the following examples.

(Example 1) (1) Synthesis of probe A single-stranded DNA probe was used as a probe capable of specifically binding to a target substance. A single-stranded nucleic acid of SEQ ID NO: 1 was synthesized using an automatic DNA synthesizer. Sequence number 1
The end of the single-stranded DNA has a thiol modifier (Thiol-Modify) at the time of synthesis by an automatic DNA synthesizer.
r) (manufactured by Glen Research) was used to introduce the mercapto (SH) group. Subsequently, usual deprotection was carried out, DNA was recovered, purified by high performance liquid chromatography, and used in the following experiments. SEQ ID NO: 15'HS- (CH2) 6-O-PO2-O-ACTGGCCGTCGTTTTACA3 '(2) Water-soluble polymer material As a water-soluble polymer material, polyvinyl alcohol (PV
A) was selected. Poval containing polyvinyl alcohol (PVA) (trade name: Poval PVA-117; Co., Ltd.)
5 g of Kuraray Co., Ltd.) was weighed and placed in a beaker. This polyvinyl alcohol has a degree of polymerization of 1700 and a degree of saponification of 9
It is 8.0 to 99.0 mol%. Pure water 495g
Dissolved in pure water by adding
% Poval aqueous solution was prepared. At the time of melting, 60 while heating to 80 ° C in a hot bath to completely dissolve
Stir for minutes. After confirming that there was no insoluble matter, filtration was performed so as to prevent nozzle clogging during spotting. A 0.22 μm membrane filter was used for filtration. The PVA aqueous solution was prepared as described above.

(3) Preparation of probe medium The single-stranded DNA probe of SEQ ID NO: 1 was added to a TE solution (10 mM T) so that the final concentration was about 40 μmol / l.
It was dissolved in ris-HCl (pH 8) / 1 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution) to prepare a single-stranded DNA probe solution (the exact concentration was calculated from the absorption intensity).

Next, a silane coupling agent (trade name: KBM-603; Shin-Etsu Chemical Co., Ltd.) containing a silane compound having an amino group (N-β (aminoethyl) γ-aminopropyltrimethoxysilane) as a probe fixing substance. 500 μmol / l aqueous solution of 3) at room temperature.
The mixture was stirred for 0 minutes to prepare an aminosilane aqueous solution. Next, a divalent reagent having a maleimide group and a hydroxysuccinimide ester group, N- (6-maleimidocapr
oyloxy) sulfosuccinimide, s
odium salt (trade name: sulfo-EMC
S; 500 μmol / l of Dojindo Laboratories Co., Ltd.)
The aqueous solution was stirred at room temperature for 5 minutes to prepare an EMCS aqueous solution. The above-mentioned aminosilane aqueous solution and EMCS aqueous solution were mixed in equal volumes and stirred for 5 minutes to obtain a probe-immobilized substance solution. The prepared probe-immobilized substance solution was used in the following experiments.

The probe solution prepared as described above was added to 10
0 μl was prepared, 100 μl of the probe-immobilized substance solution prepared as described above was added dropwise thereto, and then mixed for 5 minutes. After leaving the mixed solution for 10 minutes, 700 μl of pure water,
100 μl of an aqueous solution of PVA, which is a water-soluble polymer material prepared as described above, was added dropwise and mixed for 5 minutes. After mixing, the mixture was left for 30 minutes to prepare the probe medium.

(4) Substrate cleaning A glass substrate of 1 inch square is placed in a cassette and preheated to 60 ° C.
The glass substrate was immersed in a 1 mol / l sodium hydroxide aqueous solution heated for 10 minutes. Then, the glass substrate and the cassette were rinsed thoroughly with running pure water to wash the sodium hydroxide and remove it. After sufficiently rinsing, the glass substrate together with the cassette was immersed in pure water and ultrasonic cleaning was performed for 10 minutes. After ultrasonic cleaning, it was thoroughly rinsed in running pure water to wash and remove particles adhering to the glass substrate and the cassette. The glass substrates after washing with water were dried one by one by blowing nitrogen gas.

(5) Spotting of probe medium The probe medium prepared in (3) above was filled in an ink tank for a Bubble Jet (registered trademark) printer (BJF850; manufactured by Canon Inc.), and bubble jet (registered) was registered. (Trademark) head. The bubble jet (registered trademark) printer used here (product name: BJF
850; manufactured by Canon Inc. is modified so that printing on a flat plate is possible. Next, the glass substrate prepared in (4) above was mounted on this printer, and the probe medium was spotted on the glass substrate. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the glass substrate was 1.2 to 1.5 mm.
After the spotting was completed, the glass substrate was observed with a microscope, and it was confirmed that a matrix-like spot array was formed on the surface of the glass substrate. After completion of spotting, the glass substrate was left in a desiccator.
In this way, a probe-immobilized substrate was produced.

(6) Storage The probe-immobilized substrate prepared in (5) above was stored in a desiccator for 7 days. During this time, humidity 35% or less, temperature 2
The temperature was adjusted to 5 ° C.

(7) Blocking treatment The probe-immobilized substrate stored in (6) above was treated with 1M NaC.
It was washed with a 1/50 mM phosphate buffer (pH 7.0) solution and wetted on the probe-immobilized substrate so that uniform blocking treatment could be performed. Then, the glass substrate was immersed in a 2% bovine serum albumin aqueous solution and left for 2 hours for blocking treatment.

(8) Hybridization Treatment A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 1 described in (1) above was used as D.
A single-stranded DNA probe which was synthesized by an NA automatic synthesizer and labeled with rhodamine bound to the 5'end was obtained. The labeled single-stranded DNA was dissolved in 1 M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-treated probe-immobilized substrate obtained in (7) above was dissolved in this solution. Was dipped and subjected to a hybridization treatment at room temperature (25 ° C.) for 3 hours. Then, the probe array was washed with a 1 M NaCl / 50 mM phosphate buffer solution (pH 7.0) to wash away the single-stranded DNA probe that did not hybridize with the probe nucleic acid.
Then, after removing excess salt with pure water, the probe-immobilized substrate was dried by nitrogen blowing. Next, the fluorescence of the spot on the probe-immobilized substrate is analyzed by a fluorescence scanner (trade name: G
enePix4000B; AxonInstrumen
ts, Inc. Manufactured) was used to evaluate the fluorescence intensity. For evaluation, set the laser power to 100%, and
MT was set to 400V.

(9) Results When the fluorescence scanner evaluation results in (8) above were analyzed, the spot of the DNA probe of SEQ ID NO: 1 which was a perfect match with the labeled single-stranded DNA probe was 532 nm.
The fluorescent intensity at 15 was 15830. Further, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 61
It was 1. When the spots of each DNA probe are observed by fluorescence, each spot shape is almost circular,
Almost no difference in fluorescence intensity was observed between spots spotted with the same probe medium. Also,
The spacing between adjacent spots is almost equal, and is approximately 200
It was observed that the spots were arranged in a grid pattern at intervals of μm.

Example 2 (1) Synthesis of probe A single-stranded DNA probe was prepared in exactly the same manner as in Example 1 above, and then used in the following experiment.

(2) Water-soluble polymer material An aqueous solution of water-soluble polymer material was prepared in the same manner as in Example 1 above. As the water-soluble polymer material, POVAL containing polyvinyl alcohol (PVA) (trade name: POVAL PV
A-110; manufactured by Kuraray Co., Ltd. was used. This polyvinyl alcohol has a polymerization degree of 1000 and a saponification degree of 98.
It is 0-99.0 mol%.

(3) Adjustment of probe medium A probe solution was prepared in the same manner as in Example 1 above.

The probe solution prepared as described above was added to 10
0 μl was prepared, and 800 μl of an aqueous solution containing 7.5 wt% glycerin, 7.5 wt% urea, and 7.5 wt% thiodiglycol was added dropwise thereto. 100 μl of the water-soluble polymer material aqueous solution prepared as described above was dropped, and then mixed for 5 minutes. After mixing, the mixture was left for 30 minutes to obtain a probe medium.

(4) Cleaning of Substrate The substrate was cleaned in the same manner as in Example 1 to prepare a glass substrate.

(5) Probe immobilization treatment A silane coupling agent containing a silane compound having an amino group (N-β (aminoethyl) γ-aminopropyltrimethoxysilane) (trade name: KBM-603; Shin-Etsu Chemical Co., Ltd. ( A 1 wt% aqueous solution of Co., Ltd.) was stirred at room temperature for 30 minutes to prepare an aqueous silane coupling agent solution. Next, the glass substrate washed in (4) above was immersed in this aqueous solution for 20 minutes at room temperature (25 ° C.), and then rinsed thoroughly with running water to remove the excess aqueous solution of the silane coupling agent. The rinsed substrate was dried by dropping nitrogen gas on both sides of the substrate. Next, the substrate was baked in an oven heated to 120 ° C. for 1 hour to complete the silane coupling treatment. Then N-maleimidocaproyloxysuccinimide N-
(6-maleimidocaployloxy) su
lfosuccinimide (trade name: EMCS;
2.7 mg of Dojindo Laboratories Co., Ltd. was weighed and dissolved in a 1: 1 dimethylsulfoxide (DMSO) / ethanol solution to a final concentration of 0.3 mg / ml,
An EMCS solution was prepared. The substrate subjected to the silane coupling treatment was immersed in this EMCS solution for 30 minutes at room temperature. The substrate pulled up from the EMCS solution is sequentially washed with a mixed solvent of DMSO and ethanol and ethanol,
It was dried under a nitrogen gas atmosphere. In this way, the probe immobilization process was completed.

(6) Spotting of Probe Medium The probe medium prepared in (3) above was spotted in exactly the same manner as in Example 1 to produce a probe-immobilized substrate. After the spotting was completed, the glass substrate was observed with a microscope, and it was confirmed that a matrix-like spot array was formed on the surface of the glass substrate. After completion of spotting, the glass substrate was left in a desiccator. In this way, a probe-immobilized substrate was produced.

(7) Storage The probe-immobilized substrate prepared in (6) above was stored in a desiccator for 7 days. During this time, humidity 35% or less, temperature 2
The temperature was adjusted to 5 ° C.

(8) Blocking treatment Blocking treatment was carried out in the same manner as in Example 1 above.

(9) Hybridization treatment The hybridization treatment was carried out in the same manner as in Example 1 above. After that, the probe array was set to
Single-stranded D that did not hybridize with the probe nucleic acid after washing with Cl / 50 mM phosphate buffer (pH 7.0) solution
The NA probe was washed away. Then, after removing excess salt with pure water, the probe-immobilized substrate was dried by nitrogen blowing. Next, the fluorescence of the spot on the probe-immobilized substrate was analyzed by a fluorescence scanner (trade name: GenePix4000).
B; Axon Instruments, Inc. Manufactured) was used to evaluate the fluorescence intensity. In the evaluation, the laser power was set to 100% and the PMT was set to 400V.

(10) Results When the fluorescence scanner evaluation results in (9) above were analyzed, the spot of the DNA probe of SEQ ID NO: 1 which was a perfect match with the labeled single-stranded DNA probe was 532 nm.
The fluorescence intensity at 11520 was 11520. Further, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 65
It was 9. When the spots of each DNA probe are observed by fluorescence, each spot shape is almost circular,
Almost no difference in fluorescence intensity was observed between spots spotted with the same probe medium. Also,
The spacing between adjacent spots is almost equal, and is approximately 200
It was observed that the spots were arranged in a grid pattern at intervals of μm.

(Example 3) (1) Synthesis of probe A single-stranded DNA probe was used as a probe capable of specifically binding to a target substance. Single-stranded nucleic acids of SEQ ID NOs: 1 to 4 were synthesized using an automatic DNA synthesizer. The single-stranded nucleic acids of SEQ ID NOs: 1 to 4 are SEQ ID NO: 1 used in Example 1.
Based on the above, the one with one base changed was SEQ ID NO: 2, the one with 3 bases changed was SEQ ID NO: 3, and the one with 6 bases changed was SEQ ID NO: 4. In addition, the single-stranded DNA ends of SEQ ID NOs: 1 to 4 are Thi
The thiol (SH) group was introduced by using ol-Modifier (manufactured by Glen Research). Subsequently, normal deprotection was performed to recover the DNA, which was purified by high performance liquid chromatography and used in the following experiments. The sequences of SEQ ID NOs: 2 to 4 are shown below. SEQ ID NO: 2 5'HS- (CH2) 6-O-PO2-O-ACTGGCCGTTGTTTTACA3 'SEQ ID NO: 3 5'HS- (CH2) 6-O-PO2-O-ACTGGCCGCTTTTTTACA3' SEQ ID NO: 45'HS- (CH2) 6-O-PO2-O-ACTGGCATCTTGTTTACA3 '

(2) Water-soluble polymer material An aqueous PVA solution was prepared in exactly the same manner as in Example 1 above.

(3) Preparation of probe medium A single-stranded DNA probe solution was prepared in exactly the same manner as in Example 1 above.

Using the single-stranded DNAs of SEQ ID NOS: 1 to 4, four kinds of probe media were prepared in the same manner as the method described in (3) of Example 1 (the exact concentration is the absorption intensity. Calculated from).

(4) Substrate cleaning Substrate cleaning was performed in the same manner as in Example 1 above to prepare a glass substrate.

(5) Spotting of probe medium The four types of probe medium prepared in the above (3) were applied to a Bubble Jet (registered trademark) printer (trade name: BJF85).
0; four ink tanks for Canon Inc.) were filled with each probe medium, and each ink tank was attached to a bubble jet (registered trademark) head. The Bubble Jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) used here was modified so that printing on a flat plate was possible. Next, attach the glass substrate prepared in (4) to this printer,
The probe medium was spotted on a glass substrate. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the glass substrate is 1.2 to 1.5 mm.
Met. After the spotting was completed, the glass substrate was observed with a microscope, and it was confirmed that a matrix-like spot array was formed on the surface of the glass substrate.
After completion of spotting, the glass substrate was left in a desiccator. In this way, a probe-immobilized base material on which four types of probes were immobilized was produced.

(6) Storage The probe-immobilized substrate prepared in (5) above was stored in a desiccator for 7 days. During this time, humidity 35% or less, temperature 2
The temperature was adjusted to 5 ° C.

(7) Blocking Treatment The substrate was washed with a 1M NaCl / 50 mM phosphate buffer solution (pH 7.0) without performing the blocking treatment carried out in Example 1 above, and a uniform hybridization treatment was performed. I made it possible.

(8) Hybridization treatment The hybridization treatment was carried out in the same manner as in Example 1 above. After that, the probe array was set to
Single-stranded D that did not hybridize with the probe nucleic acid after washing with Cl / 50 mM phosphate buffer (pH 7.0) solution
The NA probe was washed away. Then, after removing excess salt with pure water, the probe-immobilized substrate was dried by nitrogen blowing. Next, the fluorescence of the spot on the probe-immobilized substrate was analyzed by a fluorescence scanner (trade name: GenePix4000).
B; Axon Instruments, Inc. Manufactured) was used to evaluate the fluorescence intensity. In the evaluation, the laser power was set to 100% and the PMT was set to 400V.

(9) Results When the fluorescence scanner evaluation results in (7) above were analyzed, it was found that the spot of the DNA probe of SEQ ID NO: 1 which was a perfect match with the labeled single-stranded DNA probe was 532 nm.
The fluorescence intensity at 15120 was 15120. On the other hand, 1
A fluorescence amount of 9030 was obtained in the spot of the DNA probe of SEQ ID NO: 2 having a base mismatch sequence. Further, in the spot of the DNA probe of SEQ ID NO: 3 having a 3-base mismatch, only a fluorescence amount less than half that of the perfect match with 3423 was obtained, and the 6-base mismatch of SEQ ID NO: 4 was obtained.
Almost no fluorescence spot was recognizable with DNA of No. 3, and the fluorescence intensity at the spot corresponding site was 973. Also, DN
It was 653 when the fluorescence intensity other than the spot part of A probe was observed. When the spots of each DNA probe were observed by fluorescence, each spot shape was almost circular, and almost no difference in fluorescence intensity was observed between the spots spotted with the same probe medium.
Further, it was observed that the intervals between the adjacent spots were almost equal, and the spots were arranged in a grid pattern at intervals of about 200 μm. From the above, it was possible to specifically detect the completely complementary single-stranded DNA on the DNA array substrate.

Example 4 (1) Synthesis of Probe A single-stranded DNA probe was used as a probe capable of specifically binding to a target substance. A single-stranded DNA probe of SEQ ID NO: 5 was synthesized using an automatic DNA synthesizer. At the end of the single-stranded DNA of SEQ ID NO: 1, an 18-mer oligomer having a 5'-terminal hydroxyl group bonded to an amino group via a phosphate group and hexamethylene was prepared and used in the following experiments. SEQ ID NO: 5 5'H2N- (CH2) 6-O-PO2-O-ACTGGCCGTCGTTTTACA3 '(2) Water-soluble polymer material As a water-soluble polymer material, polyvinyl alcohol (PV
A) was selected. Poval containing polyvinyl alcohol (PVA) (trade name: Poval PVA-217E;
5 g of Kuraray Co., Ltd. was weighed and placed in a beaker.
This polyvinyl alcohol has a degree of polymerization of 1700 and a degree of saponification of 87.0 to 89.0 mol%. Pure water 49
Dissolve it in pure water by adding 5 g to a concentration of 1.0
A wt% Poval aqueous solution was prepared. Upon dissolution, the mixture was stirred for 60 minutes while heating to 80 ° C. in a hot bath for complete dissolution. After confirming that there is no insoluble matter,
Filtration was performed so as to prevent nozzle clogging during spotting. A 0.22 μm membrane filter was used for filtration. The PVA aqueous solution was prepared as described above.

(3) Preparation of probe medium The single-stranded DNA probe of SEQ ID NO: 5 was added to a TE solution (10 mM T) so that the final concentration was about 40 μmol / l.
It was dissolved in ris-HCl (pH 8) / 1 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution) to prepare a single-stranded DNA probe solution (the exact concentration was calculated from the absorption intensity).

Next, a silane coupling agent (trade name: KBM-403; manufactured by Shin-Etsu Chemical Co., Ltd.) containing a silane compound having an epoxy group (γ-glycidoxypropyltrimethoxysilane) as a probe fixing substance was used. Fifty
A 0 μmol / l aqueous solution was stirred at room temperature for 30 minutes to obtain a probe-immobilized substance solution. The prepared probe-immobilized substance solution was used in the following experiments.

The probe solution prepared as described above was added to 10
0 μl was prepared, 100 μl of the probe-immobilized substance solution prepared as described above was added dropwise thereto, and then mixed for 5 minutes. After leaving the mixed solution for 10 minutes, 700 μl of pure water,
100 μl of an aqueous solution of PVA, which is a water-soluble polymer material prepared as described above, was added dropwise and mixed for 5 minutes. After mixing, the mixture was left for 30 minutes to prepare the probe medium.

(4) Substrate cleaning Substrate cleaning was performed in the same manner as in Example 1 above to prepare a glass substrate.

(5) Spotting of Probe Medium A probe-immobilized substrate was prepared by spotting the probe medium onto the substrate in the same manner as in Example 1 above.

(6) Storage The probe-immobilized substrate prepared in (5) above was stored in a desiccator for 7 days. During this time, humidity 35% or less, temperature 2
The temperature was adjusted to 5 ° C.

(7) Blocking Treatment The probe-immobilized substrate stored in (6) above was treated with 1M NaC.
It was washed with a 1/50 mM phosphate buffer (pH 7.0) solution and wetted on the probe-immobilized substrate so that uniform blocking treatment could be performed. Then, the glass substrate was immersed in a 2% bovine serum albumin aqueous solution and left for 2 hours for blocking treatment.

(8) Hybridization Treatment A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 5 described in (1) above was used as D.
A single-stranded DNA probe which was synthesized by an NA automatic synthesizer and labeled with rhodamine bound to the 5'end was obtained. The labeled single-stranded DNA was dissolved in 1 M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-treated probe-immobilized substrate obtained in (7) above was dissolved in this solution. Was dipped and subjected to a hybridization treatment at room temperature (25 ° C.) for 3 hours. Then, the probe array was washed with a 1 M NaCl / 50 mM phosphate buffer solution (pH 7.0) to wash away the single-stranded DNA probe that did not hybridize with the probe nucleic acid.
Then, after removing excess salt with pure water, the probe-immobilized substrate was dried by nitrogen blowing. Next, the fluorescence of the spot on the probe-immobilized substrate is analyzed by a fluorescence scanner (trade name: G
enePix4000B; AxonInstrumen
ts, Inc. Manufactured) was used to evaluate the fluorescence intensity. For evaluation, set the laser power to 100%, and
MT was set to 400V.

(9) Results When the fluorescence scanner evaluation results in (8) above were analyzed, it was found that the spot of the DNA probe of SEQ ID NO: 5 which was a perfect match with the labeled single-stranded DNA probe was 532 nm.
The fluorescence intensity was 17110. Moreover, when the fluorescence intensity other than the spot portion of the DNA probe was observed, 73
It was 1. When the spots of each DNA probe are observed by fluorescence, each spot shape is almost circular,
Almost no difference in fluorescence intensity was observed between spots spotted with the same probe medium. Also,
The spacing between adjacent spots is almost equal, and is approximately 200
It was observed that the spots were arranged in a grid pattern at intervals of μm.

(Example 5) (1) Synthesis of probe Single-stranded DN of SEQ ID NO: 5 was prepared in exactly the same manner as in Example 4 above.
After preparing the A probe, it was used in the following experiment.

(2) Water-soluble polymer material An aqueous PVA solution was prepared in exactly the same manner as in Example 1 above.

(3) High boiling point solvent As the high boiling point solvent, propylene glycol (1,2-propanediol; manufactured by Kishida Chemical Co., Ltd.) was selected. Propylene glycol (5.2 g) was weighed, and isopropyl alcohol (2-propanol; Kishida Chemical Co., Ltd.)
(Manufactured by the company) was weighed and mixed. Isopropyl alcohol was mixed in consideration of extraction and mixing properties when preparing the probe medium. The high boiling point solvent liquid was prepared as described above.

(4) Preparation of probe medium The single-stranded DNA probe of SEQ ID NO: 5 was dispensed and dried at a concentration of 17.53 nmol per microtube, and a microtube containing the single-stranded DNA probe was prepared. I had it.

Prepare one microtube containing the single-stranded DNA probe dispensed as described above and add pure water to it.
After dropping 0 μl, the mixture was stirred for 3 minutes to sufficiently dissolve the DNA. A silane coupling agent (trade name: KB) containing a silane compound having an epoxy group (γ-glycidoxypropyltrimethoxysilane) as a probe fixing substance
20 μl of M-403; manufactured by Shin-Etsu Chemical Co., Ltd. was dropped, and then mixed for 10 minutes. After allowing the mixed solution to stand for 20 minutes, the PVA aqueous solution 10 which is the above water-soluble polymer material
0 μl and 1400 μl of high boiling point solvent solution were added dropwise and mixed for 5 minutes. Finally, 430 μl of pure water was added dropwise, mixed for 5 minutes, and then allowed to stand for 30 minutes. In this way, the probe medium was prepared.

(5) Substrate Cleaning Substrate cleaning was performed in the same manner as in Example 1 above to prepare a glass substrate.

(6) Spotting of Probe Medium A probe-immobilized substrate was prepared by spotting a probe medium onto a substrate in the same manner as in Example 1 above.

(7) Storage The probe-immobilized substrate prepared in (6) above was stored in a desiccator for 7 days. During this time, humidity 35% or less, temperature 2
The temperature was adjusted to 5 ° C.

Further, in order to confirm the dry storage characteristics as a probe-immobilized substrate, the probe-immobilized substrate was dried on a hot plate at 80 ° C. for 5 minutes and then similarly stored in a desiccator for 7 days.

(8) Hybridization Treatment A single-stranded DNA probe having a nucleotide sequence complementary to the single-stranded DNA probe of SEQ ID NO: 5 described in (1) above was used as D.
A single-stranded DNA probe which was synthesized by an NA automatic synthesizer and labeled with rhodamine bound to the 5'end was obtained. This labeled single-stranded DNA was dissolved in 1 M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the probe-immobilized substrate was immersed in this solution and placed in an incubator (45 ° C.). Hybridization was carried out for 2 hours. After that, the probe array was set to 1M Na.
Single-stranded D that did not hybridize with the probe nucleic acid after washing with Cl / 50 mM phosphate buffer (pH 7.0) solution
The NA probe was washed away. Then, after removing excess salt with pure water, the probe-immobilized substrate was dried by nitrogen blowing. Next, the fluorescence of the spot on the probe-immobilized substrate was analyzed by a fluorescence scanner (trade name: GenePix4000).
B; Axon Instruments, Inc. Manufactured) was used to evaluate the fluorescence intensity. In the evaluation, the laser power was set to 100% and the PMT was set to 400V.

(9) Results When the fluorescence scanner evaluation results in (8) above were analyzed, it was found that in the probe-immobilized substrate stored for 7 days in a desiccator, SEQ ID NO: 5 which is a perfect match with the labeled single-stranded DNA probe. 532n in the DNA probe spot of
The fluorescence intensity at m was 12970. Moreover, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 1
It was 31. On the other hand, in the probe-immobilized substrate dried on a hot plate at 80 ° C. for 5 minutes, the DN of SEQ ID NO: 5 which is a perfect match with the labeled single-stranded DNA probe was used.
At spot A, the fluorescence intensity at 532 nm is 13540.
Met. In addition, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 96. When the spots of each DNA probe were observed by fluorescence, each spot shape was almost circular, and almost no difference in fluorescence intensity was observed between the spots spotted with the same probe medium. Further, it was observed that the intervals between the adjacent spots were almost equal, and the spots were arranged in a grid pattern at intervals of about 200 μm.

(Example 6) (1) Synthesis of probe Single-stranded DN of SEQ ID NO: 5 was prepared in exactly the same manner as in Example 4 above.
After preparing the A probe, it was used in the following experiment.

(2) Water-soluble polymer material A PVA aqueous solution was prepared in exactly the same manner as in Example 1 above.

(3) High boiling solvent Diethylene glycol (1,2-propanediol; manufactured by Kishida Chemical Co., Ltd.) was selected as the high boiling solvent. Propylene glycol (5.6 g) was weighed, and isopropyl alcohol (2-propanol; Kishida Chemical Co., Ltd.) was added thereto.
(Manufactured by the company) was weighed and mixed. Isopropyl alcohol was mixed in consideration of extraction and mixing properties when preparing the probe medium. The high boiling point solvent liquid was prepared as described above.

(4) Preparation of probe medium The single-stranded DNA probe of SEQ ID NO: 5 was dispensed and dried at a rate of 17.53 nmol per microtube, and a microtube containing the single-stranded DNA probe was prepared. I had it.

Prepare one microtube containing the single-stranded DNA probe dispensed as described above, and add pure water to it.
After dropping 0 μl, the mixture was stirred for 3 minutes to sufficiently dissolve the DNA. A silane coupling agent (trade name: KBE-9007; manufactured by Shin-Etsu Chemical Co., Ltd.) containing a silane compound having an isocyanate group (γ-isocyanatopropyltriethoxysilane) as a probe fixing substance was added thereto.
After dripping μl, the mixture was mixed for 10 minutes. 2 mixed solution
After standing for 0 minutes, the above water-soluble polymer material PVA
100 μl of an aqueous solution and 1400 μl of a high boiling point solvent solution were added dropwise and mixed for 5 minutes. Finally, 430 μl of pure water was added dropwise, mixed for 5 minutes, and then allowed to stand for 30 minutes. In this way, the probe medium was prepared.

(5) Substrate cleaning Substrate cleaning was performed in the same manner as in Example 1 above to prepare a glass substrate.

(6) Spotting of Probe Medium In the same manner as in Example 1 above, spotting of the probe medium onto the substrate was performed to prepare a probe-immobilized substrate.

(7) Storage The probe-immobilized substrate prepared in (6) above was stored in a desiccator for 7 days. During this time, humidity 35% or less, temperature 2
The temperature was adjusted to 5 ° C.

Further, in order to confirm the dry storage characteristics of the probe-immobilized substrate, the probe-immobilized substrate was dried on a hot plate at 80 ° C. for 5 minutes and then similarly stored in a desiccator for 7 days.

(8) Hybridization Treatment A single-stranded DNA probe having a nucleotide sequence complementary to the single-stranded DNA probe of SEQ ID NO: 5 described in (1) above was used as D.
A single-stranded DNA probe which was synthesized by an NA automatic synthesizer and labeled with rhodamine bound to the 5'end was obtained. This labeled single-stranded DNA was dissolved in 1 M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the probe-immobilized substrate was immersed in this solution and placed in an incubator (45 ° C.). Hybridization was carried out for 2 hours. After that, the probe array was set to 1M Na.
Single-stranded D that did not hybridize with the probe nucleic acid after washing with Cl / 50 mM phosphate buffer (pH 7.0) solution
The NA probe was washed away. Then, after removing excess salt with pure water, the probe-immobilized substrate was dried by nitrogen blowing. Next, the fluorescence of the spot on the probe-immobilized substrate was analyzed by a fluorescence scanner (trade name: GenePix4000).
B; Axon Instruments, Inc. Manufactured) was used to evaluate the fluorescence intensity. In the evaluation, the laser power was set to 100% and the PMT was set to 400V.

(9) Results When the fluorescence scanner evaluation results in (8) above were analyzed, it was found that in the probe-immobilized substrate stored for 7 days in a desiccator, SEQ ID NO: 5 which was a perfect match with the labeled single-stranded DNA probe. 532n in the DNA probe spot of
The fluorescence intensity at m was 17790. Moreover, when the fluorescence intensity other than the spot portion of the DNA probe was observed, 8
It was 9. On the other hand, in the probe-immobilized base material dried on a hot plate at 80 ° C. for 5 minutes, the DNA of SEQ ID NO: 5 which is a perfect match with the labeled single-stranded DNA probe.
In the spot, the fluorescence intensity at 532 nm was 18340. In addition, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 81. When the spots of each DNA probe were observed by fluorescence, each spot shape was almost circular, and almost no difference in fluorescence intensity was observed between the spots spotted with the same probe medium. Further, it was observed that the intervals between the adjacent spots were almost equal, and the spots were arranged in a grid pattern at intervals of about 200 μm.

Comparative Example 1 (1) Synthesis of probe Single-stranded DN of SEQ ID NO: 1 was prepared in exactly the same manner as in Example 1 above.
After preparing the A probe, it was used in the following experiment.

(2) Water-Soluble Polymer Material In order to confirm the effect of the water-soluble polymer material, a probe-immobilized substrate was prepared without using the water-soluble polymer material.

(3) Preparation of probe medium Single strand D of SEQ ID NO: 1 was prepared in exactly the same manner as in Example 1 above.
An NA probe solution was prepared (the exact concentration was calculated from the absorption intensity). Then, in the same manner as in Example 1, a probe-immobilized substance solution containing an aminosilane and a divalent reagent was prepared. The prepared probe-immobilized substance solution was used in the following experiments.

The probe solution prepared as described above was added to 10
0 μl was prepared, 100 μl of the probe-immobilized substance solution prepared as described above was added dropwise thereto, and then mixed for 5 minutes. After leaving the mixed solution for 10 minutes, 800 μl of pure water was dropped and mixed for 5 minutes. After mixing, leave for 30 minutes,
The probe medium was adjusted.

(4) Substrate Cleaning Substrate cleaning was performed in the same manner as in Example 1 above to prepare a glass substrate.

(5) Spotting of Probe Medium A probe-immobilized substrate was prepared by spotting the probe medium on the substrate in the same manner as in Example 1.

(6) Storage The probe-immobilized substrate prepared in (5) above was stored in a desiccator for 7 days. During this time, humidity 35% or less, temperature 2
The temperature was adjusted to 5 ° C.

(7) Blocking treatment The probe-immobilized substrate stored in (6) above was treated with 1M NaC.
It was washed with a 1/50 mM phosphate buffer (pH 7.0) solution and wetted on the probe-immobilized substrate so that uniform blocking treatment could be performed. Then, the glass substrate was immersed in a 2% bovine serum albumin aqueous solution and left for 2 hours for blocking treatment.

(8) Hybridization Treatment A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 1 described in (1) above was used as D.
A single-stranded DNA probe which was synthesized by an NA automatic synthesizer and labeled with rhodamine bound to the 5'end was obtained. The labeled single-stranded DNA was dissolved in 1 M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-treated probe-immobilized substrate obtained in (7) above was dissolved in this solution. Was dipped and subjected to a hybridization treatment at room temperature (25 ° C.) for 3 hours. Then, the probe array was washed with a 1 M NaCl / 50 mM phosphate buffer solution (pH 7.0) to wash away the single-stranded DNA probe that did not hybridize with the probe nucleic acid.
Then, after removing excess salt with pure water, the probe-immobilized substrate was dried by nitrogen blowing. Next, the fluorescence of the spot on the probe-immobilized substrate is analyzed by a fluorescence scanner (trade name: G
enePix4000B; AxonInstrumen
ts, Inc. Manufactured) was used to evaluate the fluorescence intensity. For evaluation, set the laser power to 100%, and
MT was set to 400V.

(9) Results When the results of the fluorescence scanner evaluation in (8) above were analyzed, the spot of the DNA probe of SEQ ID NO: 1 which was a perfect match with the labeled single-stranded DNA probe was 532 nm.
The fluorescence intensity at was 1427. Further, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 697.
Met. When the spots of each DNA probe were observed by fluorescence, each spot shape was almost circular, and almost no difference in fluorescence intensity was observed between the spots spotted with the same probe medium. In addition, the space between adjacent spots is almost equal, and is approximately 200 μm.
It was observed that the spots were arranged in a grid pattern at intervals of m. As described above, when the probe medium containing no water-soluble polymer material was used, the fluorescence intensity at the spot portion in the perfect match was not obtained, and the detection could not be effectively performed.

Comparative Example 2 (1) Synthesis of Probe Single-stranded DN of SEQ ID NO: 1 was prepared in exactly the same manner as in Example 1 above.
After preparing the A probe, it was used in the following experiment.

(2) Water-Soluble Polymer Material In order to confirm the effect of the water-soluble polymer material, a probe-immobilized substrate was prepared without using the water-soluble polymer material.

(3) Preparation of probe medium Single strand D of SEQ ID NO: 1 was prepared in exactly the same manner as in Example 1 above.
An NA probe solution was prepared (the exact concentration was calculated from the absorption intensity). The prepared probe solution was used in the following experiments.

The probe solution prepared as described above was added to 10
0 μl was prepared, and 900 μl of an aqueous solution containing 7.5 wt% glycerin, 7.5 wt% urea, and 7.5 wt% ethylene glycol was added dropwise thereto. After mixing for 5 minutes,
After standing for 30 minutes, the probe medium was adjusted.

(4) Substrate Cleaning Substrate cleaning was performed in the same manner as in Example 1 above to prepare a glass substrate.

(5) Probe immobilization treatment A probe immobilization treatment was carried out on a glass substrate washed in the same manner as in Example 2 above.

(6) Spotting of Probe Medium The probe medium prepared in (3) above was spotted in exactly the same manner as in Example 1 to produce a probe-immobilized substrate. After the spotting was completed, the glass substrate was observed with a microscope, and it was confirmed that a matrix-like spot array was formed on the surface of the glass substrate. After completion of spotting, the glass substrate was left in a desiccator. In this way, a probe-immobilized substrate was produced.

(7) Storage The probe-immobilized substrate prepared in (6) above was stored in a desiccator for 7 days. During this time, humidity 35% or less, temperature 2
The temperature was adjusted to 5 ° C.

(8) Blocking Treatment Blocking treatment was carried out in the same manner as in Example 1 above.

(9) Hybridization treatment The hybridization treatment was performed in exactly the same manner as in Example 1 above. After that, the probe array was set to 1M Na.
Single-stranded D that did not hybridize with the probe nucleic acid after washing with Cl / 50 mM phosphate buffer (pH 7.0) solution
The NA probe was washed away. Then, after removing excess salt with pure water, the probe-immobilized substrate was dried by nitrogen blowing. Next, the fluorescence of the spot on the probe-immobilized substrate was analyzed by a fluorescence scanner (trade name: GenePix4000).
B; Axon Instruments, Inc. Manufactured) was used to evaluate the fluorescence intensity. In the evaluation, the laser power was set to 100% and the PMT was set to 400V.

(10) Results When the results of the fluorescence scanner evaluation in (9) above were analyzed, it was found that the spot of the DNA probe of SEQ ID NO: 1 which was a perfect match with the labeled single-stranded DNA probe was 532 nm.
The fluorescence intensity at 1527 was 1527. Moreover, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 792.
Met. When the spots of each DNA probe were observed by fluorescence, each spot shape was almost circular, and almost no difference in fluorescence intensity was observed between the spots spotted with the same probe medium. In addition, the space between adjacent spots is almost equal, and is approximately 200 μm.
It was observed that the spots were arranged in a grid pattern at intervals of m.

Comparative Example 3 (1) Synthesis of Probe Single-stranded DN of SEQ ID NO: 5 was prepared in exactly the same manner as in Example 4 above.
After preparing the A probe, it was used in the following experiment.

(2) Water-Soluble Polymer Material In order to confirm the effect of the water-soluble polymer material, a probe-immobilized substrate was prepared without using the water-soluble polymer material.

(3) High boiling solvent In order to confirm the effect of the high boiling solvent, a probe-immobilized substrate was prepared without using the high boiling solvent.

(4) Preparation of probe medium Single strand D of SEQ ID NO: 5 was prepared in exactly the same manner as in Example 4 above.
An NA probe solution was prepared (the exact concentration was calculated from the absorption intensity). PVA which is the water-soluble polymer material of (2) above
Pure water was added instead of the solution, and isopropyl alcohol was added dropwise as a substitute solvent for the high boiling point solvent in (3) above and mixed.
The prepared probe solution was used in the following experiments.

(5) Substrate Cleaning Substrate cleaning was performed in the same manner as in Example 1 above to prepare a glass substrate.

(6) Probe immobilization treatment A probe immobilization treatment was carried out on the glass substrate washed in the same manner as in Example 1 above.

(7) Spotting of Probe Medium The probe medium prepared in (4) above was spotted in the same manner as in Example 1 to prepare a probe-immobilized substrate. After the spotting was completed, the glass substrate was observed with a microscope, and it was confirmed that a matrix-like spot array was formed on the surface of the glass substrate. After completion of spotting, the glass substrate was left in a desiccator. In this way, a probe-immobilized substrate was produced.

Further, some of the probe-immobilized substrates were subjected to hot plate treatment at 80 ° C. for 5 minutes and then left standing in a desiccator.

(8) Storage The probe-immobilized substrate prepared in (7) above was stored in a desiccator for 7 days. During this time, humidity 35% or less, temperature 2
The temperature was adjusted to 5 ° C.

(9) Hybridization treatment The hybridization treatment was performed in the same manner as in Example 1 above. After that, the probe array was set to 1M Na.
Single-stranded D that did not hybridize with the probe nucleic acid after washing with Cl / 50 mM phosphate buffer (pH 7.0) solution
The NA probe was washed away. Then, after removing excess salt with pure water, the probe-immobilized substrate was dried by nitrogen blowing. Next, the fluorescence of the spot on the probe-immobilized substrate was analyzed by a fluorescence scanner (trade name: GenePix4000).
B; Axon Instruments, Inc. Manufactured) was used to evaluate the fluorescence intensity. In the evaluation, the laser power was set to 100% and the PMT was set to 400V.

(10) Results When the fluorescence scanner evaluation results in (9) above were analyzed, it was found that in the probe-immobilized substrate stored in a desiccator for 7 days, SEQ ID NO: 5 which is a perfect match with the labeled single-stranded DNA probe. 532n in the DNA probe spot of
The fluorescence intensity at m was 4276. Moreover, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 89
Met. On the other hand, in the probe-immobilized substrate dried at 80 ° C. for 5 minutes on the hot plate, the labeled single-stranded D
In the DNA spot of SEQ ID NO: 5 which was a perfect match with the NA probe, the fluorescence intensity at 532 nm was 1286. In addition, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 81. When the spots of each DNA probe were observed by fluorescence, each spot shape was almost circular, and almost no difference in fluorescence intensity was observed between the spots spotted with the same probe medium. Further, it was observed that the intervals between the adjacent spots were almost equal, and the spots were arranged in a grid pattern at intervals of about 200 μm.

(Effects of Embodiments and Examples) As described above, according to the embodiments and examples of the present invention, by incorporating the probe and the water-soluble polymer material into the probe medium, the probe The probe can be efficiently and stably immobilized on the substrate simply by applying the medium onto the substrate, and the target substance can be effectively detected by the probe even after storage. .
Further, by producing a probe-immobilized substrate by using this method, not only the probe-immobilized substrate can be produced in a small number of steps, but also a probe-immobilized substrate having high stability and good storage stability can be obtained. It can be manufactured simply.

Further, according to the present invention, the probe medium can be easily observed even after being spotted on the substrate and dried. By observing and confirming the shape, state, etc. of the spot on which the probe medium is spotted, it is possible to check whether the probe medium was spotted well. Therefore, when various types of probes are spotted on one substrate, it is possible to determine whether the spots are independent and whether contamination has occurred. By being able to confirm the contamination, it is possible to reliably detect the target substance.

Further, according to the present invention, it is possible to obtain a probe array capable of more accurately inspecting more information on a target substance even from a small amount of a sample, and by using the probe array, the presence of the target substance in the sample can be obtained. Whether or not to do so can be determined more accurately and quickly. Similarly, by using this probe array, the structure of the target substance in the sample can be more accurately and rapidly specified.

[0145]

According to the present invention, the probe can be efficiently and stably immobilized on the substrate.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Makoto Kameyama             Kyano, 3-30-2 Shimomaruko, Ota-ku, Tokyo             Within the corporation F-term (reference) 4B024 AA11 CA09 HA14                 4B029 AA07 BB20 CC03 FA15

Claims (9)

[Claims] 1. A probe medium comprising a probe capable of specifically binding to a target substance, at least a water-soluble polymer material and a high boiling point solvent. 2. The probe medium according to claim 1, wherein the water-soluble polymer material is polyvinyl alcohol. 3. The probe medium according to claim 1, wherein the high boiling point solvent is a glycol high boiling point organic solvent. 4. The probe medium according to claim 1, wherein the probe is a DNA probe. 5. A probe fixing method for fixing by fixing the probe medium according to claim 1 on a substrate by a spotting method. 6. A DNA array in which the probe of the probe-immobilized substrate produced by the method of immobilizing a probe according to claim 5 is a DNA probe. 7. A detection method in which the probe medium according to claim 1 is fixed by being applied onto a substrate by a spotting method, and a target substance is detected using the obtained probe-immobilized substrate. 8. The probe according to claim 1, the water-soluble polymer material, and the high-boiling solvent are housed in respective containers,
A probe medium that is mixed when immobilized on a substrate. 9. A probe medium, wherein the water-soluble polymer material and the high boiling point solvent according to claim 1 improve the storage characteristics of a probe-immobilized substrate.