JP2001066305A - Liquid composition for emitting probe by ink jet method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently and accurately spot a trace amount of a probe on a solid phase without damaging the probe by incorporating the probe, an urea, a glycerin, a thiodiglycol and an acetylene alcohol represented by specific formula. SOLUTION: A liquid 107 containing a nucleic acid probe is moved to an emitting port 119 to form a meniscus 121 at a predetermined pressure. Here, when an electric signal is applied to electrodes 111-1, 111-2 of a substrate part 117, a foaming area 123 is abruptly heated, and foam is generated in the liquid 107 brought in contact with the area 123. The meniscus 121 is emitted by its pressure, the liquid 107 is discharged from a emitting port 119, and liquid droplets 104 are flown to a surface of a solid phase 103. The emitted liquid 107 is preferably a liquid containing a glycerin, a urea, a thiodiglycol and an acetylene alcohol represented by formula (wherein R1 to R4 are each an alkyl group, as for m, n, m=0 and n=0 or 1<=m+n<=30, and in the case of m+n=1, m or n is 0).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for spotting a probe on a solid phase, a probe array, a method for producing the same, a method for detecting a target single-stranded nucleic acid using the probe array, and a base sequence of the target single-stranded nucleic acid. Related to the specification method.

[0002]

2. Description of the Related Art As one of techniques capable of rapidly and accurately determining a nucleotide sequence of a nucleic acid, detecting a target nucleic acid in a sample, and identifying various bacteria, for example, a substance capable of specifically binding to the target nucleic acid, The use of a so-called probe array in which a large number of probes are arranged on a solid phase has been proposed.

[0003] As a general method for producing such a probe array, for example, as described in EP 373203 (EP 0373203 B1), (1) a method of synthesizing a nucleic acid probe on a solid phase, And (2) a method of supplying a previously synthesized nucleic acid probe onto a solid phase, and the like. Prior art disclosing the details of the above method (1) includes, for example, US Pat.
No. 783 (US Pat. No. 5,405,783).

As the method (2), for example, US Pat. No. 5,601,980 (US Pat.
0) and "Science", 270
Vol., Page 467, (1995) discloses a method for arranging cDNAs in an array using micropipetting.

In the above method (1), since a nucleic acid probe is directly synthesized on a solid phase, there is no need to synthesize a nucleic acid probe in advance. However, it is difficult to purify the probe nucleic acid synthesized on the solid phase. The accuracy of nucleic acid base sequencing using a probe array, detection of a target nucleic acid in a sample, and the like greatly depends on the accuracy of the nucleic acid probe base sequence. Therefore, in the method (1), as a method for producing a higher-quality probe array, further improvement is required for improving the accuracy of the nucleic acid probe.

On the other hand, the above method (2) requires a step of synthesizing the nucleic acid probe before immobilizing the nucleic acid probe on the solid phase, but purifies the nucleic acid probe prior to binding to the solid phase. it can. For this reason, at the present stage, the method (2) is considered to be more preferable than the method (1) as a method for producing a higher quality probe array.

However, the problem of the above method (2) lies in a method of spotting a nucleic acid probe at a high density on a solid phase. For example, when a nucleic acid base sequence is determined using a probe array, it is preferable to arrange as many types of nucleic acid probes as possible on a solid phase. In order to efficiently detect gene mutations, it is preferable to arrange nucleic acid probes having sequences corresponding to the respective mutations on a solid phase. Further, in detecting a target nucleic acid in a sample, or detecting a mutation or a deletion of a gene, it is preferable that collection of a sample from a subject, specifically, collection of blood or the like be kept as small as possible. It is preferable that information on as many base sequences as possible can be obtained from the specimen. Considering these points, it is preferable that, for example, 10,000 or more nucleic acid probes are arranged in one inch square in the probe array.

[0008]

As a result of various studies under such circumstances, the present inventors have found that it is possible to spot a probe at extremely high density using an ink jet ejection technique. Invented the invention.

An object of the present invention is to provide a method for spotting an extremely small amount of a probe efficiently and accurately on a solid phase without damaging the probe.

Another object of the present invention is to provide a probe array capable of more accurately examining more information on a nucleic acid from a small amount of a sample.

It is still another object of the present invention to provide a method for efficiently producing a probe array in which a large number of probes are bound on a solid phase without damaging the probes.

It is still another object of the present invention to provide a method for efficiently detecting a target substance that may be contained in a sample.

Still another object of the present invention is to provide a method for specifying the structure of a target substance, which can obtain information on the structure of the target substance from a small amount of a sample.

[0014]

A liquid composition for discharging a probe according to an embodiment of the present invention, which can achieve the above object, by an ink jet method specifically binds to a target substance. A liquid composition for discharging a possible probe by an inkjet method, comprising a probe, urea, glycerin, thiodiglycol, and acetylene alcohol represented by the following general formula (I). Is:

[0015]

Embedded image

(Wherein R ₁ , R ₂ , R ₃ and R ₄ represent an alkyl group, m and n each represent an integer, and m =
0 and n = 0 or 1 ≦ m + n ≦ 30, and m
When + n = 1, m or n is 0. By using the spotting method according to the above aspect, a probe can be accurately and efficiently provided on a solid phase, and a probe array can be efficiently manufactured.

Further, the probe array according to an embodiment of the present invention is characterized in that probe spots independent of each other are provided at a plurality of sites on the surface of the solid phase at a density of 10,000 or more per square inch. It is assumed that. Since the probe array according to the above aspect has spots at a very high density, much information can be obtained even from a small amount of sample.

The method for producing a probe array according to one embodiment of the present invention is directed to a probe having a spot containing a probe capable of specifically binding to a target substance independently at a plurality of positions on a solid phase surface. A method for producing an array, comprising a step of supplying a liquid containing the probe to a predetermined position on the surface of the solid phase using an ink-jet method, and attaching the liquid to the predetermined position. According to this aspect, it is possible to efficiently manufacture a probe array in which spots are densely arranged without damaging the probe.

The above object can be achieved.
The method for detecting a target substance according to one embodiment of the present invention includes, as a plurality of spots independent of each other, a probe that specifically binds to a target substance that may be contained in a sample on a solid phase. A method of contacting each sample of the probe array with the sample, detecting a reaction product of the target substance and the probe on the solid phase to detect the presence or absence of the target substance in the sample, Are formed by spotting a liquid containing the probe on a solid phase by an ink-jet method. According to this aspect, the target substance can be efficiently detected.

Further, the above object can be achieved.
The method for specifying the structure of a target substance according to one embodiment of the present invention is a method for specifying the structure of a target substance contained in a sample, which specifically binds to the specific substance on a solid phase surface. A step of preparing a probe array having spots of probes to be performed; a step of bringing the sample into contact with the spots; and a step of detecting the binding between the target substance and the probes. By using this embodiment, the structure of the target substance in a small amount of a sample, for example, when the target substance is a single-stranded nucleic acid, can be used to efficiently specify the base sequence thereof.

US Pat. No. 5,601,980 recognizes that it is not appropriate to use a conventional ink jet technique for spotting a nucleic acid probe. That is, the third column in the second column of US Pat.
Lines 1 to 52 describe that it is not appropriate to use the ink jet printer technology for discharging a small amount of ink by a pressure wave, because the pressure wave for discharging the ink is caused by a sudden increase in the ink temperature. This raises the risk of causing an excessive temperature rise, possibly damaging the nucleic acid probe, and the danger of scattering of ink at the time of ejection causing contamination between spots of adjacent nucleic acid probes. Further, in US Pat. No. 5,601,980, a droplet of a liquid containing a nucleic acid probe is formed at the tip of a micropipette while monitoring the size of the droplet by using gas pressure, and pressure is applied when the droplet reaches a predetermined size. And manufacturing a probe array by supplying the droplets onto a solid phase.

In US Pat. No. 5,474,796, a hydrophobic and hydrophilic matrix is formed on the surface of a solid phase, and four types of bases are added to the hydrophilic portion of the matrix by a piezoelectric impulse jet pump device.
t Pump Apparatus) to produce an oligonucleotide array by sequential ejection, and a method for determining a base sequence of a target nucleic acid using the oligonucleotide array.

However, none of these prior arts discloses a technique of discharging a nucleic acid probe having a base sequence of a predetermined length in advance by using an ink jet technique to arrange nucleic acid probes with high density and accuracy. It has not been.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS (Outline of Probe Array Manufacturing Method) FIG.
2 and FIG. 2 are schematic illustrations of a method for producing a probe array, for example, a nucleic acid probe array according to the present invention. In FIG. 1, reference numeral 101 denotes a liquid supply system (nozzle) which holds a probe as a discharge liquid, for example, a liquid containing a nucleic acid probe, and 103 denotes a solid phase (for example, a transparent glass plate or the like) to which the nucleic acid probe is to be bound. ) And 105 are bubble jet heads each of which is a kind of an ink jet head and has a mechanism for applying thermal energy to the liquid and discharging the liquid. 104 is a bubble jet (registered trademark) head 105
This is a liquid containing a nucleic acid probe discharged from. FIG. 2 is a sectional view taken along line AA of the bubble jet head 105 of FIG. 1. In FIG. 2, reference numeral 105 denotes a bubble jet head; 107, a liquid containing a nucleic acid probe to be discharged;
Reference numeral 117 denotes a substrate portion having a heat generating portion for applying discharge energy to the liquid. The substrate portion 117 includes a protective film 109 formed of silicon oxide or the like, electrodes 111-1 and 111-2 formed of aluminum or the like, a heating resistor layer 113 formed of nichrome or the like, and a heat storage layer 1.
15 and a support 116 made of alumina or the like having good heat dissipation.

The liquid 107 containing the nucleic acid probe has a discharge orifice (discharge port) 119, and forms a meniscus 121 by a predetermined pressure. Here electrode 11
When an electric signal is applied to 1-1 and 111-2, an area (foaming area) indicated by 123 rapidly generates heat, bubbles are generated in the liquid 107 in contact with the area, and the meniscus is discharged by the pressure, and the orifice is discharged. The liquid 107 is discharged from 119 and flies toward the surface of the solid phase 103. The amount of liquid that can be ejected using a bubble jet head having such a structure varies depending on the size of the nozzle and the like.
It can be controlled to about 50 picoliters,
This is extremely effective as a means for arranging nucleic acid probes at high density. (Relation between ejected liquid and solid phase) (Spot diameter on solid phase) Density of nucleic acid probe on solid phase is as described above (for example, 10000 for each inch)
(The upper limit is about 1 × 10 ⁶ ), the spot diameter of each nucleic acid probe is, for example, 20 to 1
It is preferably about 00 μm, and the spots are preferably independent of each other. Such spots are determined by the characteristics of the liquid ejected from the bubble jet head, the surface characteristics of the solid phase to which the liquid adheres, and the like.

(Characteristics of Discharged Liquid) As the liquid for discharge,
The liquid can be ejected from the bubble jet head, and the liquid ejected from the head lands at a desired position on the solid phase. Further, the nucleic acid probe is not damaged in a mixed state with the nucleic acid probe and at the time of ejection. Any liquid can be used.

From the viewpoint of dischargeability from the bubble jet head, the liquid preferably has, for example, a viscosity of 1 to 15 cps and a surface tension of 30 dyn / cm or more. The viscosity is 1-5 cps and the surface tension is 3
When it is 0 to 50 dyn / cm, the impact position on the solid phase becomes extremely accurate, and it is particularly preferably used.

Next, in consideration of the ink jet discharge characteristics of the liquid and the stability of the nucleic acid probe in the liquid and at the time of the bubble jet discharge, for example, 2 mer to 500
It is preferable that a nucleic acid probe of 0 mer, particularly 2 mer to 60 mer is contained at a concentration of 0.05 to 500 µM, particularly 2 to 50 µM.

(Composition of Discharged Liquid) The composition of the liquid discharged from the bubble jet head has a substantial effect on the nucleic acid probe when mixed with the nucleic acid probe as described above and when discharged from the bubble jet head. The liquid composition is not particularly limited as long as the liquid composition satisfies the preferable conditions and the liquid composition that can be normally ejected to the solid phase using a bubble jet head satisfies the preferable conditions, for example, glycerin, urea, and thiol. A liquid containing diglycol or ethylene glycol, isopropyl alcohol and acetylene alcohol represented by the following formula (I) is preferable.

[0030]

Embedded image

(In the above formula (I), R ₁ , R ₂ , R ₃ and R ₄
Represents an alkyl group, specifically, for example, a linear or branched alkyl group having 1 to 4 carbon atoms, m and n each represent an integer, and m = 0 and n = 0, or 1 ≦ m + n
When ≦ 30 and m + n = 1, m or n is 0. More specifically, 5-10 wt% of urea and 5% of glycerin
-10% by weight, 5-10% by weight of thiodiglycol, and 0.1% by weight of the acetylene alcohol represented by the above formula (I).
A liquid containing 02 to 5 wt%, more preferably 0.5 to 1 wt% is suitably used.

When this liquid is used, when a liquid containing a nucleic acid probe is ejected from a bubble jet head and deposited on a solid phase, the spot has a circular shape, and the ejected area does not expand. Even when a nucleic acid probe is spotted at a high density, the connection with an adjacent spot can be effectively suppressed. Further, no alteration of the nucleic acid probe spotted on the solid phase is observed. The properties of the liquid used for producing the nucleic acid probe array of the present invention are not limited to those described above. For example, a well-like structure that prevents a liquid applied on a solid phase by a bubble jet head on the solid phase surface from spreading on the solid phase and mixing with an adjacent spot is used. In the case where it is provided, it can be used even if the viscosity and surface tension of the liquid, as well as the base length and concentration of the nucleic acid probe are out of the above ranges.

(Types of functional groups of solid phase and nucleic acid) Effective for stopping the spot of nucleic acid probe attached on the solid phase at a further limited position to more reliably prevent contamination with adjacent spots. As an effective means for firmly binding a nucleic acid probe to a solid phase, there is a method of binding functional groups capable of reacting with each other to both the nucleic acid probe and the solid phase.

(SH group and maleimide group) Preferred examples include, for example, an example using a combination of a maleimide group and a thiol (-SH) group. That is, by attaching a thiol (-SH) group to the end of the nucleic acid probe and treating the solid phase surface so as to have a maleimide group,
The thiol group of the nucleic acid probe supplied to the solid phase surface reacts with the maleimide group of the solid phase surface to immobilize the nucleic acid probe, so that a spot of the nucleic acid probe can be formed at a predetermined position on the solid phase. . Particularly when a nucleic acid probe having a thiol group at the end dissolved in a liquid having the above composition is applied to the surface of a solid phase in which a maleimide group is introduced using a bubble jet head, the nucleic acid probe solution is extremely small on the solid phase. Form spots. As a result, a small spot of the nucleic acid probe can be formed at a predetermined position on the surface of the solid phase. In this case, for example, a well composed of a hydrophilic and hydrophobic matrix is formed on the solid phase surface,
It is not necessary to provide a configuration for preventing connection between spots in advance.

For example, a liquid containing a nucleic acid probe having a base length of 18 mer at a concentration of 8 μM and having its viscosity and surface tension adjusted to fall within the above-mentioned ranges is supplied to a bubble jet printer (trade name: BJC620; manufactured by Canon Inc.). However, when the distance between the solid phase and the nozzle of the bubble jet head is set to about 1.2 to 1.5 mm by using a plate which has been modified so as to allow printing on a flat plate and the nozzle is ejected (the ejection amount is about 24 Picoliter), about 70-100μ in diameter on the solid phase
m spots could be formed, and no spots (hereinafter, referred to as “satellite spots”) derived from droplets when the liquid landed on the solid phase surface were visually observed. The reaction between the maleimide group on the solid phase and the SH group at the end of the nucleic acid probe is completed in about 30 minutes at room temperature (25 ° C.), depending on the conditions of the liquid to be discharged. This time is shorter than the case where a piezo jet head is used for discharging the liquid. Although the reason is not clear, in the bubble jet method, the temperature of the liquid containing the nucleic acid probe in the head increases due to the principle, and as a result, the reaction efficiency between the maleimide group and the thiol group increases, and the reaction time is shortened. Conceivable.

When a combination of a maleimide group and a thiol group is used, it is preferable that the solution containing the nucleic acid probe contains thiodiglycol. The thiol group forms a disulfide bond (-S- under neutral and weak alkaline conditions).
S-) may be formed to form a dimer. However, the addition of thiodiglycol can prevent a decrease in the reactivity between the thiol group and the maleimide group due to dimer formation.

Various methods can be used to introduce a maleimide group onto the surface of the solid phase. For example, a glass substrate is reacted with an aminosilane coupling agent, and then the amino group is reacted with an N-type compound represented by the following structural formula. (6-maleimidocaproyloxy) succinimide (N- (6-Maleimidocaproylox)
y) Reagent containing succinimide) (EMCS reagent: Doji)
(manufactured by n company).

[0038]

Embedded image

The nucleic acid probe to which the thiol group is bonded can be used as a 5′-terminal reagent when automatically synthesizing DNA using, for example, an automatic DNA synthesizer.
The compound can be synthesized by using differentiator C6 (manufactured by Glen Research), and can be obtained by purification by high performance liquid chromatography after a usual deprotection reaction.

(Amino group and epoxy group) Examples of combinations of functional groups used for immobilization include, in addition to the combination of thiol group and maleimide group described above, for example, epoxy group (on solid phase) and amino group (nucleic acid). Probe end). The introduction of an epoxy group to the surface of the solid phase can be performed, for example, by applying polyglycidyl methacrylate having an epoxy group to a solid surface made of a resin, or applying a silane coupling agent having an epoxy group to a glass solid surface. A method of reacting with glass is exemplified.

As described above, when a functional group which reacts with each other to form a covalent bond on the surface of the solid phase and the end of the single-stranded nucleic acid probe is introduced, the nucleic acid probe and the solid phase are more strongly bound to each other. Is done. In addition, since the binding site of the nucleic acid probe to the solid phase can always be at the end, the state of the nucleic acid probe at each spot can be made uniform.
As a result, the conditions for hybridization between the nucleic acid probe and the target nucleic acid at each spot are made uniform, and it is considered that detection of the target nucleic acid and identification of the base sequence with further improved accuracy can be performed. In addition, covalently bonding a nucleic acid probe having a functional group at its end to a solid phase has a greater sequence and length than a nucleic acid probe immobilized on a solid phase by non-covalent bonding (eg, electrostatic bonding). A probe array can be prepared quantitatively without causing a difference in the amount of probe DNA bound due to the difference. Furthermore, since the entire nucleotide sequence of the nucleic acid contributes to the hybridization reaction, the efficiency of the hybridization reaction can be significantly increased. Further, for example, an alkylene group having about 1 to 7 carbon atoms is introduced as a linker between a portion of the single-stranded nucleic acid probe involved in hybridization with the target nucleic acid and a functional group involved in the reaction with the solid phase. May be. This makes it possible to provide a predetermined distance between the surface of the solid phase and the nucleic acid probe when the nucleic acid probe is bound to the solid phase, and is expected to further improve the reaction efficiency between the nucleic acid probe and the target nucleic acid. it can.

(Manufacturing Method of Array) Next, one of the presently most preferable embodiments of the manufacturing method of the probe array according to the present invention will be described. First, 7.5 wt% of glycerin and 7.5 wt% of urea are used as a liquid for dispersing the nucleic acid probe.
%, Thiodiglycol 7.5 wt%, the above general formula (I)
Acetylene alcohol having a structure represented by the following formula (for example, trade name: acetylenol EH; Kawaken Fine Chemical Co., Ltd.)
A liquid containing 1 wt% (produced by the company) is prepared. Next, a thiol group is bonded to the terminal, and the length is, for example, 2 to 5000 me.
A single-stranded nucleic acid probe of about r, especially about 2 to 60 mer is synthesized using an automatic DNA synthesizer. Next, the nucleic acid probe is added to the liquid, for example, at a concentration of 0.05 to 50.
In the range of 0 μM, particularly 2 to 50 μM, the viscosity of the liquid is 1 to 15 cps, especially 1 to 5 cps, and the surface tension is 30 dyn / cm or more, particularly 30 to 50 dyn / cm.
To obtain a liquid for ejection. Then, the ejection liquid is filled into, for example, a nozzle of a bubble jet head. Further, a maleimide group is introduced into the surface of the solid phase in accordance with the above method. Then, the distance between the surface of the solid phase and the bubble jet head, to which the maleimide group of the solid phase is bonded, and the nozzle surface of the bubble jet head are 1.2 to 2.0.
The bubble jet head is driven close to about 1.5 mm to discharge the liquid. Here, it is preferable that the ejection conditions are set to a print pattern such that the spots on the solid phase are not connected to each other. For example, when the resolution of the bubble jet head used for spotting is 360 × 720 dpi, the ejection is performed once in the direction of 360 dpi, then the ejection is performed twice, and in the direction of 720 dpi, the ejection is performed once and the ejection is performed five times. When spotting, the space between each spot is about 100
μm, and contamination with adjacent spots can be sufficiently prevented.

Next, the reaction between the maleimide group on the solid phase and the thiol group of the nucleic acid probe in the liquid proceeds, and the solid phase is allowed to stand, for example, in a humidified chamber until the nucleic acid probe is securely fixed to the solid phase. As described above, for example, about 30 minutes at room temperature (about 25 ° C.) is sufficient. Thereafter, unreacted nucleic acid probes on the solid phase are washed away to obtain a nucleic acid probe array.

Here, using this nucleic acid probe array,
For example, the detection accuracy (S /
For the purpose of improving the N ratio), after the nucleic acid probe is immobilized on the solid phase surface, blocking is performed so that the nucleic acid probe non-binding portion of the solid phase does not bind to the target nucleic acid or the like contained in the sample. Is preferred. The blocking can be performed, for example, by immersing the solid phase in a 2% bovine serum albumin aqueous solution, for example, for about 2 hours, or by decomposing a maleimide group that is not bonded to the nucleic acid probe on the surface of the solid phase. For example, DTT (dithiothreitol), β-mercaptoethanol, or the like can be used. However, in view of the effect of preventing the adsorption of the labeled DNA,
Bovine serum albumin aqueous solution is most suitable. This blocking step may be performed as needed.For example, when the supply of the sample to the probe array is limited to each spot and the sample does not substantially adhere to a site other than the spot. May not be performed. Also, the blocking step can be omitted when a well is formed in advance on the solid phase and a portion other than the well is processed so that the nucleic acid probe is hardly attached thereto.

The probe array prepared in this manner may have a plurality of spots containing the same nucleic acid probe, for example, or may have a plurality of spots each containing a different kind of nucleic acid probe, depending on its use. It may be configured as follows. The probe array in which nucleic acid probes are arranged at a high density by such a method is then used for detection of a target single-stranded nucleic acid, identification of a base sequence, and the like. For example, when used for detection of a target single-stranded nucleic acid having a known base sequence that may be contained in a sample, the base sequence has a base sequence complementary to the base sequence of the target single-stranded nucleic acid. Using a single-stranded nucleic acid as a probe, to prepare a probe array in which a plurality of spots containing the probe are arranged on a solid phase, to each spot of the probe array,
After supplying the sample and placing it under conditions such that the target single-stranded nucleic acid hybridizes with the nucleic acid probe, the presence or absence of a 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. When used for specifying the base sequence of the target single-stranded nucleic acid contained in the sample, a plurality of candidates for the base sequence of the target single-stranded nucleic acid are set, and each candidate is complementary to the base sequence group. A single-stranded nucleic acid having a specific base sequence is spotted to the solid phase as a probe. Next, a sample is supplied to each spot, and the conditions are such that the target single-stranded nucleic acid and the nucleic acid probe are hybridized. Then, the presence or absence of a hybrid in each spot is detected by a known method such as fluorescence detection. I do. Thereby, the base sequence of the target single-stranded nucleic acid can be specified. Other uses of the probe array according to the present invention include, for example, 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.

(Type of Inkjet Head) In the above description, only the configuration in which the nucleic acid probe is applied to the solid phase by the bubble jet head has been described. In the present invention, the vibration pressure of the piezo element is used. It is also possible to use a piezo jet head for discharging the liquid in the nozzle. However, when a bubble jet head is used as described above, the binding reaction to the solid phase is completed in a short time,
In addition, since the secondary structure of DNA is also eliminated by heat, the efficiency of the subsequent hybridization reaction can be increased, and the bubble jet head is an ink jet head more preferably used in the present invention.

Further, a plurality of spots may be simultaneously formed on the solid phase by using an ink jet head having a plurality of heads so that the nucleic acid probes contained between two or more spots are different. (PNA / DNA) The present invention has been described using a nucleic acid probe as an example of the probe. Examples of nucleic acid probes include deoxyribonucleic acid (DNA) probes, ribonucleic acid (RNA) probes, and peptide nucleic acids (PNA)
It includes a probe. PNA is the four bases contained in DNA (adenine, guanine, thymine, cytosine)
Is a synthetic oligonucleotide that binds not to the sugar-phosphate ester main chain but to the peptide main chain and has a structure as shown in the following formula (II).

[0048]

Embedded image

(In the formula, “Base” represents 4 which constitutes DNA.
Different bases (adenine, cytosine, thymine, guanine)
Is shown. P represents the base length of PNA. PNA can be synthesized, for example, by a method known as a tBOC-type solid-phase synthesis method or an Fmoc-type solid-phase synthesis method. PNA has stronger resistance to enzymes such as nucleases and proteases than natural oligonucleotides such as DNA and RNA, and is stable with little or no enzymatic cleavage in serum. In addition, since it does not have a sugar moiety or a phosphate group, it is hardly affected by the ionic strength of the solution. Furthermore, since there is no electrostatic repulsion, the hybrid of PNA and the target single-stranded nucleic acid is better than the hybrid of the DNA probe and the target single-stranded nucleic acid or the hybrid of the RNA probe and the target single-stranded nucleic acid. It is also considered to have excellent thermal stability. From these characteristics, it is promising as a probe used for detection of a target nucleic acid or determination of a base sequence. The above-described method for producing a nucleic acid probe array according to the present invention is also effective when a PNA probe is applied as a nucleic acid probe. Can be manufactured. Specifically, for example, PNA
As a method for increasing the density of the probe array by stopping the probe at a limited position on the solid phase, as in the case of the DNA probe and the RNA probe, the reactivity of the terminal of the PNA probe and the surface of the solid phase with each other is increased. A method of introducing a functional group can be used, and one preferable combination of reactive groups is a combination of a thiol group (PNA terminal) and a maleimide group (solid surface) as described above. . P
The introduction of a thiol group to the NA terminal is, for example, to introduce a cysteine (CH (NH ₂ ) (COOH) CH ₂ SH) group containing a thiol group at the N terminal of the PNA probe (corresponding to the 5 ′ terminal of DNA). Is achieved in. Introduction of cysteine to the N-terminal of the PNA probe can be performed, for example, by reacting the N-terminal amino group of the PNA probe with the carboxyl group of cysteine. The N-terminal amino group of the PNA probe and the carboxyl group of a suitable linker having an amino group and a carboxyl group such as N ₂ H (CH ₂ ) ₂ O (CH ₂ ) ₂ OCH ₂ COOH And then reacting the amino group of the linker with the carboxyl group of cysteine to bind cysteine to the N-terminal of the PNA probe via the linker. When a bonding group to a solid phase is introduced via a linker in this manner, the reaction site of the PNA probe with the target substance can be separated from the solid phase by a predetermined distance, and the hybridization efficiency can be further improved. Be expected.

Depending on the base length of PNA, the solubility in water may be lower than that of DNA having the same base length. When preparing a liquid for ink jet discharge, PNA is preliminarily prepared with trifluoroacetic acid (for example, 0.1%). (1% by weight trifluoroacetic acid aqueous solution) or the like, and then it is preferable to use the above-described various solvents to adjust the characteristics to be suitable for inkjet discharge. In particular, dissolving in trifluoroacetic acid prevents denaturation to cystine by oxidation of the thiol group in the cysteine residue at the PNA terminal,
It is preferable to further improve the reaction efficiency between the thiol group of A and the maleimide group on the surface of the solid phase. The reaction time between the thiol group introduced into the terminal of the DNA probe or the RNA probe and the maleimide group on the solid phase surface is 30 minutes (when a bubble jet head is used) as described above. Even when a bubble jet head is used, the reaction is preferably performed for about 2 hours.

The probe is not limited to a nucleic acid probe, but may be a substance capable of specifically binding to a target substance in a sample to be detected or analyzed, for example, a ligand capable of specifically binding to a receptor, or a ligand capable of specifically binding to a ligand. Oligopeptides or polypeptides that can bind to a receptor that can bind, an oligopeptide or polypeptide having a specific amino acid sequence, and a protein (eg, an antibody, an antigen, an enzyme, or the like) can be used as a probe. In this case, each of the cysteine residues contained in the protein
H groups can be utilized in the reaction.

As described above, according to the probe array manufacturing method including the step of supplying the probe solution to the solid phase by using the inkjet discharge process, the probe array can be formed extremely easily. In particular, when a functional group is introduced into each of the nucleic acid probe and the solid phase surface so that a covalent bond is formed, the solid phase surface does not have a well or the like in advance, ie, is substantially flat and has a surface characteristic. Even when a solid phase having uniform (e.g., wettability with water) is used, adjacent spots are not connected. As a result, a nucleic acid probe array in which nucleic acid probes are arranged with high precision and high density can be manufactured extremely efficiently and at low cost.

This does not exclude the use of a solid phase having a well on the surface in the present invention. For example, when a light-impermeable matrix pattern (hereinafter referred to as a “black matrix”) is formed in advance between wells to which a probe solution is supplied, hybridization of the probe and the target substance on the solid phase is performed. In such a case, the detection accuracy (SN ratio) in the case where the application is optically detected (for example, the detection of fluorescence) can be further improved. When a matrix having a low affinity for the probe solution is provided between adjacent wells, the probe solution can be smoothly transferred to the desired well even if there is some displacement in supplying the probe solution to the well. Can be supplied. For the purpose of utilizing such an effect, a solid phase having a well on the surface may be used. Hereinafter, a solid phase having a matrix on the surface, a method for producing the solid phase, and a method for using the solid phase in this embodiment will be described.

FIG. 5 shows an example of the probe array in this embodiment. FIG. 5A is a plan view, and FIG. 5B is a BB cross-sectional view thereof. This probe array is a solid phase 1
Recesses (wells) 12 arranged in a matrix on
Matrix pattern 12 having a frame structure formed with 7
5 is provided. The wells 127 isolated from each other by the matrix 125 (projection) are provided as through-holes (cut-out portions) in the matrix pattern. Is in an exposed state. The exposed surface of the solid phase 103 forms a surface that can be bonded to the probe, and the probe (not shown) is fixed in a predetermined concave portion.

The material for forming the matrix pattern is to optically detect a reaction product between the probe and the target substance.
When the method of measuring and detecting the fluorescence emitted from the reactant is used, a material having light-shielding properties is desirable in consideration of the improvement in detection sensitivity, S / N ratio, and reliability. Examples of such a material include a metal (chromium, aluminum, gold, etc.) and a black resin. As the black resin, acrylic, polycarbonate, polystyrene, polyethylene,
Resins such as polyimide, acrylic acid monomer, urethane acrylate, and the like, and photosensitive resins such as photoresists containing a black dye or a black pigment may be used. Specific examples of the photosensitive resin include, for example, UV resist, DE
An EP-UV resist, an ultraviolet curable resin, or the like can be used. As UV resist, cyclized polyisoprene-
Negative resists such as aromatic bis azide resists, phenol resin-aromatic azide compound resists, and positive resists such as novolak resin-diazonaphthoquinone resists can be used.

As the DEEP-UV resist, as a positive resist, for example, polymethyl methacrylate,
Radiation-decomposable polymer resists such as polymethylene sulfone, polyhexafluorobutyl methacrylate, polymethyl isopropenyl ketone, and poly 1-trimethylsilylpropyne bromide, and dissolution inhibitor-based resists such as o-nitrobenzyl cholic acid. As a negative resist, polyvinylphenol-
3,3′-diazidodiphenyl sulfone, polyglycidyl methacrylate, and the like can be given.

The UV-curable resin may be one selected from benzophenone and its substituted derivatives, benzoin and its substituted derivatives, acetophenone and its substituted derivatives, and oxime compounds such as benzyl. 2-10 photopolymerization initiators
Examples thereof include polyester acrylate, epoxy acrylate, and urethane diacrylate, which are contained in an amount of about weight%. As the black pigment, carbon black or a black organic pigment can be used.

When the reaction between the probe and the target substance is not optically detected, or when the light from the matrix does not affect the optical detection of the reactant, a non-matrix pattern forming material is used. The use of a light-shielding material is not hindered at all.

Next, as one method for forming a matrix pattern using the above-mentioned materials, a resin is coated on the substrate surface or a photoresist is coated on a metal, and after patterning, the resin is patterned by a process such as etching. Method. In the case of a photosensitive resin, patterning can be performed by exposing and developing the resin itself by a photolithography process using a photomask and, if necessary, curing the resin.

When the matrix 125 is made of resin, the surface of the matrix 125 becomes hydrophobic. This configuration is preferable when an aqueous solution is used as the solution containing the probe to be supplied to the well. That is, even when the probe solution is supplied to the well using the inkjet method with some displacement, the probe solution is supplied to the desired well extremely smoothly. Further, even when different types of probes are supplied simultaneously between adjacent wells, it becomes possible to prevent cross-contamination between different probe solutions supplied between these wells.

Usually, a probe solution of a biological substance such as a peptide or a nucleic acid is often an aqueous solution, and in such a case, the structure in which the matrix pattern is water-repellent can be suitably used.

Next, a description will be given of a method of making the bottom surface of the well (the exposed portion of the solid surface) connectable to the probe. The functional group held on the bottom of the well differs depending on the combination with the functional group supported on the probe. For example, when a nucleic acid probe having a thiol group introduced at the terminal is used as a probe, the thiol group of the nucleic acid probe supplied to the well can be converted into a maleimide on the solid surface by introducing a maleimide group on the solid phase surface as described above. The covalent bond is formed with the group, and the nucleic acid probe is immobilized on the solid surface. Similarly, for a nucleic acid probe having an amino group at the end of the nucleic acid probe, it is preferable to introduce an epoxy group on the surface of the solid phase. As another combination of such functional groups, for example, for a nucleic acid probe having a carboxyl group (by introducing a succinimide derivative to the end of a nucleic acid probe), introduction of an amino group to the surface of a solid phase is preferable. Although the combination of the amino group and the epoxy group has a poor fixability on the solid phase when the nucleic acid probe solution is ejected by the inkjet ejection method as compared with the combination of the thiol group and the maleimide group, Is negligible when provided.

As described above, when a glass plate is used as the solid phase, the surface of the glass plate is first treated with an alkali such as potassium hydroxide or sodium hydroxide. Hydroxyl group (silanol group)
Is exposed to the surface, and then a silane compound having an amino group introduced therein (eg, N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane) or a silane compound having an epoxy group introduced therein (eg, γ-glycidoxy) The reaction can be carried out by reacting a silane coupling agent containing propyltrimethoxysilane with hydroxyl groups on the surface of the glass plate. The maleimide group is formed on the surface of the glass plate by introducing an amino group into the surface of the glass plate by the above method and then reacting the amino group with N-maleimidocaproyloxysuccinimide or succiimidyl-4- (maleimidophenyl) butyrate. Can be introduced.

The structures of N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane, γ-glycidoxypropyltrimethoxysilane and succiimidyl-4- (maleimidophenyl) butyrate are as follows. N-β (aminoethyl) -γ-aminopropyltrimethoxysilane: (CH ₃ O) ₂ SiC ₃ H ₆ NHC ₂ H ₄ NH
₂ γ-glycidoxypropyltrimethoxysilane:

[0065]

Embedded image

Succinimidyl-4- (maleimidophenyl) butyrate:

[0067]

Embedded image

When an epoxy group is introduced to the surface of the solid phase in the above-described surface treatment of the solid phase, the unreacted epoxy group is ring-opened using an ethanolamine aqueous solution after binding the epoxy group to the probe. By changing to a hydroxyl group, the bottom surface of the well can be made hydrophilic. This operation is preferable when an aqueous solvent containing a target substance specifically reacting with the probe is supplied to the well to which the probe has been bound.

When a resin substrate is used as the solid phase, for example, Organic Thin Films and Surface, Vol. 20, Academ
A hydroxyl group, a carboxyl group, an amino group, or the like can be introduced into the resin substrate surface by the method described in Chapter 5 of IC Press. Alternatively, after introducing a hydroxyl group by this method, it is also possible to introduce an amino group or an epoxy group using a silane compound having an amino group or an epoxy group in the same manner as in the case of the above-described glass plate, or further introduce a maleimide group. It is. The introduction of the functional group into the solid phase may be performed before the formation of the matrix on the surface of the solid phase, or may be performed after the formation of the matrix. Before the formation of the matrix, the reaction solution necessary for introducing the functional group may be supplied to the solid phase surface by a method such as spin coating or dip coating on the solid phase surface. The reaction solution may be supplied to the well.

As a method of bonding a probe to a resin substrate, for example, as described in JP-A-60-015560, a surface of a resin substrate is oxidized to introduce a hydroxyl group, and then an amino group is added. A method of reacting a silane coupling agent containing a silane compound having the same with the hydroxyl group to introduce an amino group, and reacting the amino group with a functional group of a probe.

In the case where the substrate after the pretreatment is hydrophilic, the other relatively water-repellent material for forming the matrix pattern may be the resin matrix forming resin previously extended. it can. If further water repellency is required, a water repellent can be added to the matrix material. Further, when the matrix pattern is formed of a photosensitive resin such as a photoresist, it is possible to impart more water repellency to the matrix pattern by performing post-baking under appropriate conditions after exposure and development.

Up to this point, the case where the probe solution is hydrophilic has been described, but if the probe solution is lipophilic, the reverse process may be performed.

The size and shape of the wells of the matrix pattern are determined by the size of the substrate, the size of the entire array finally manufactured, the number of types of probes constituting the array, or
The method can be appropriately selected depending on a method for forming a matrix pattern, a method for supplying a probe solution or the like to the matrix pattern gap, a detection method, and the like.

The shape may be various shapes such as a rectangle, various polygons, a circle, an ellipse, in addition to a square cross section of a plane parallel to the substrate shown in FIG.

When considering the number of reaction species and the size of the entire array, the maximum width of the well is 300 μm.
μm or less is desirable. For example, as shown in FIG. 5, when the cross section of the well in the direction parallel to the substrate is a square, the length of one side can be 200 μm or less.
Further, when the well has a rectangular shape, its long side is set to 20.
When the diameter is 0 μm or less and the diameter is circular, the diameter is more preferably 200 μm or less. The lower limit of the size can be, for example, about 1 μm.

The arrangement of the wells can be changed as needed as required, such as an arrangement in which the wells are arranged at equal intervals in the vertical direction in the plan view as shown in FIG. is there.

The distance between adjacent wells is determined by, for example, cross-contamination even when a slight displacement occurs between the discharge position and the well to be supplied when the probe solution is supplied to the well by the inkjet method. It is preferable to set the interval so as not to cause it. In consideration of the size of the entire array, cross contamination, and operability in supplying various solutions, the distance between adjacent wells is one of the longest width of the well. / 2 to 2 times.

For example, when the well has a square shape, the size of the substrate is set to a size suitable for automating operations such as probe fixing, sample supply and detection (1 inch × 1 inch).
Inches or 1 cm × 1 cm), 100 × 100 or 1000 × 1 from the necessity of sufficiently fulfilling the function of combinatorial chemistry.
Since it is desirable that there are 000 or more types of probes, it is desirable that the side of the square shape of the well is 1 to 200 μm and the distance between adjacent wells is 200 μm or less in consideration of the size of the matrix itself.

The thickness of the matrix (height from the surface of the solid phase) is determined in consideration of the method of forming the matrix pattern, the volume of the well, the amount of the probe solution to be supplied, and the like. It is preferable that That is, by adopting such a thickness, for example, when a probe solution is supplied to each well using an inkjet discharge method, the characteristics of the probe solution in relation to the inkjet discharge conditions, the probe solution is placed on the solid surface. Even when it is difficult to form a small spot, the probe solution can be stopped at a predetermined position on the solid phase, and cross contamination can be prevented very effectively. Can be.

The volume of the well at the upper limit of the above-mentioned desirable range is 200 μm × 200 μm × 20 μm, that is, 800 pl. Also, with this size, if the distance between adjacent wells (x in FIG. 1) is also 200 μm, a well density of 625 / cm ² can be obtained. That is, an array having a well density of 10 ² / cm ² or more as an order is obtained. Further, when the well has a square shape with a side of 5 μm, the distance between adjacent wells is also 5 μm, and the thickness of the matrix pattern is 4 μm, the volume of the well is 0.1 pl and the number is 1,000,000.
Pieces / cm ² . Since the patterning of 5 μm × 5 μm × 4 μm is a realistic size in the current fine processing technology, an array having a well density of 10 ⁶ / cm ² or more can be included in the scope of the present invention.

In this embodiment, the probe solution or
When the amount of the liquid to be reacted with the probe to be supplied to the well is, for example, approximately the same as the volume of the well, from the above calculation, it is approximately 0.1 picoliter (pl) to 1 nanoliter (nl). Becomes Also, when the matrix is made incompatible with the supplied solution, depending on the liquid type,
The surface tension allows an amount of liquid exceeding the volume of the well to be retained at the top of the well opening. In such a case, for example, a liquid volume 10 times to several tens times that of the well can be supplied and held. That is, several pl to several tens nl of liquid are supplied. In either case, such a small amount of liquid can be supplied to the well using a general microdispenser or micropipette.However, an ink jet method that can supply the positional accuracy and the supply amount accuracy well is used. Preferably, the probe solution is used to supply the well to the well. Ink-jet printing discharges ink with high-precision positioning in the order of μm, which is extremely suitable for supplying a solution to a well. In addition, since the amount of ink to be ejected is generally several pl to several tens nl, it can be said that this is also suitable for supplying the solution to the well.

According to this embodiment, the spread of the droplet is quantitatively controlled by the reaction between the nucleic acid probe and the solid phase surface and the well, and even if there is some disturbance in the ejection direction, the area including the well is not affected. If the droplets adhere to the matrix, the portions of the droplets that touch the matrix are repelled because the matrix is incompatible with the ejection liquid, and are smoothly accommodated in the wells.

The ink jet method used in the present invention is not particularly limited. For example, a piezo jet method or a bubble jet method utilizing thermal foaming can be used.

As the material that can be used as the solid phase 103 in the present invention, any material can be used as long as it can introduce various functional groups as described above into the surface of the solid phase. Those capable of forming a matrix are preferred. When a reaction product of the probe and the target substance is optically detected, and when a detection system is provided via a solid phase, the solid phase is preferably an optically transparent solid phase. Examples of such a material include glass containing synthetic quartz, fused quartz, and the like, silicon, acrylic resin, polycarbonate resin, polystyrene resin, and vinyl chloride resin. When the optical detection of the reaction product is performed without using a solid phase, an optically black solid phase is preferably used, and a resin substrate or the like containing a black dye or pigment such as carbon black is used. .

In the present invention, a solution of a substance to be reacted is supplied to these probe arrays, and the reaction is performed under appropriate reaction conditions. When it is necessary to supply different solutions of the substances to be reacted to the individual wells, supply at least one solution in which at least one substance to be reacted with the probe is dissolved, to each of the plurality of wells of the probe array. I do. In this case, as described above, if the supplied solution has an affinity for the wells to which the probes of the already formed probe array are bound and does not have an affinity for the matrix pattern, the supply region is changed. It is possible to supply a limited amount of liquid without cross contamination. Many of the biologically-related substances, such as the substances shown in Table 1, are water-soluble. In this case, the wells are hydrophilic and the matrix pattern is water-repellent. In addition, as described above, the use of the ink jet method for the supply of these substances to be reacted makes it possible to quantitatively supply a small amount of liquid.

In the present invention, since the amount of the probe solution supplied for binding to the substrate or the amount of the substance to be reacted is very small, both reaction conditions are determined by evaporation and air diffusion of the supplied solution. It is desirable to include conditions to prevent

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 (Method for producing nucleic acid probe array using bubble jet printer and evaluation of the probe array) (1) Substrate washing A 1-inch square glass plate was placed in a rack, and was placed in a detergent for ultrasonic cleaning overnight. Soaked. Thereafter, ultrasonic cleaning was performed in a detergent for 20 minutes, and then the detergent was removed by water washing. After rinsing with distilled water, sonication was further performed for 20 minutes in a container containing distilled water. Next, the glass plate was immersed in a 1 N sodium hydroxide solution preheated to 80 ° C. for 10 minutes. Subsequently, washing with water and washing with distilled water were performed to prepare a glass plate for a probe array.

(2) Surface Treatment A silane coupling agent containing a silane compound having an amino group bonded thereto (N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane) (trade name: KBM603; Shin-Etsu Chemical Co., Ltd.) ) Was stirred at room temperature for 2 hours to hydrolyze the methoxy group in the molecule of the silane compound. Next, the substrate obtained in the above (1) was immersed in this solution at room temperature (25 ° C.) for 20 minutes, pulled up, and dried by spraying nitrogen gas on both sides of the glass plate. Next, the glass plate was baked in an oven heated to 120 ° C. for 1 hour to complete the silane coupling treatment, and amino groups were introduced on the substrate surface. Subsequently, N-maleimidocaproyloxysuccinimide (N- (6-Maleimidocaproyloxy) succini
mide; manufactured by Dojin Co., Ltd. (hereinafter abbreviated as EMCS) to 2.7
mg was weighed, and an EMCS solution was prepared by dissolving it in a 1: 1 solution of dimethyl sulfoxide (DMSO) / ethanol so that the final concentration was 0.3 mg / ml. The glass plate that had been subjected to the silane coupling treatment was immersed in this EMCS solution at room temperature for 2 hours, and the amino groups carried on the surface of the glass plate and the carboxyl groups of the EMCS solution were reacted by the silane coupling treatment. In this state, a maleimide group derived from EMCS exists on the surface of the glass plate. The glass plate pulled up from the EMCS solution is DMSO
After washing sequentially with a mixed solvent of ethanol and ethanol and ethanol, it was dried under a nitrogen gas atmosphere.

(3) Synthesis of Probe DNA A single-stranded nucleic acid of SEQ ID NO: 1 was synthesized using an automatic DNA synthesizer. In addition, DNA is added to the end of the single-stranded DNA of SEQ ID NO: 1.
When synthesizing with an automatic synthesizer, the thiol modifier (Th
iol-Modifier) (Glen Research (Gle
nResearch) was used to introduce a thiol (SH) group. Subsequently, normal deprotection is performed and D
The NA was recovered, purified by high performance liquid chromatography, and used for the following experiments. SEQ ID NO: ^{15 '} HS- (CH ₂ ) ₆ -O-PO ₂ -O-ACTGGCCGTCGTTTTACA ^3' (4) DNA ejection by BJ printer and binding to substrate Final concentration of single-stranded DNA of SEQ ID NO: 1 Is about 400m
g / ml of TE solution (10 mM Tris-
HCl (pH 8) / 1 mM EDTA aqueous solution) to prepare a single-stranded DNA solution (the exact concentration was calculated from the absorption intensity).

Glycerin 7.5 wt%, urea 7.5 wt%
%, Thiodiglycol 7.5 wt%, and acetylene alcohol represented by the general formula (I) (trade name: acetylenol EH; manufactured by Kawaken Fine Chemical Co., Ltd.) 1
An aqueous solution containing wt% is prepared and added to the above DNA solution.
The final concentration of single-stranded DNA was adjusted to 8 μM. The surface tension of this liquid is in the range of 30 to 50 dyne / cm, and the viscosity is 1.8 cps (E-type viscometer:
Tokyo Keiki Co., Ltd.). This liquid was filled in an ink tank for a bubble jet printer (trade name: BJC620; manufactured by Canon Inc.) and attached to a bubble jet head. The bubble jet printer used here (trade name: BJC620; manufactured by Canon Inc.)
Has been modified so that it can be printed on a flat plate.
This bubble jet printer is 360x720d
Printing is possible at a resolution of pi. Next, the glass plate treated in the above (2) was attached to this printer, and the liquid containing the probe nucleic acid was spotted on the glass plate. Here, the distance between the liquid ejection surface of the bubble jet head and the liquid attachment surface of the glass plate was 1.2 to 1.5 mm. The conditions of the spotting were set such that two spot discharges were performed after one spotting in the 360 dpi direction, and five spot discharges were performed after one spotting in the 720 dpi direction. After the completion of the spotting, the glass plate was allowed to stand in a humidified chamber for 30 minutes to react a maleimide group on the surface of the glass plate with a thiol group at the end of the nucleic acid probe. The discharge amount of the DNA probe solution per one discharge operation of the printer was about 24 pl.

(5) Blocking reaction After the reaction between the maleimide group and the thiol group was completed, the glass plate was washed with a 1 M NaCl / 50 mM phosphate buffer (pH 7.0).
After washing with the solution, the liquid containing DNA on the surface of the glass plate was completely washed away. Next, the glass plate was immersed in a 2% bovine serum albumin aqueous solution and left for 2 hours to perform a blocking reaction.

(6) Hybridization reaction A single-stranded DNA having a base sequence complementary to the DNA of SEQ ID NO: 1 was synthesized by an automatic DNA synthesizer, and a single-stranded DNA was labeled by binding rhodamine to the 5 ′ end. DNA was obtained. This labeled single-stranded DNA is dissolved in 1 M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-treated probe array obtained in (5) above is immersed in this solution. Then, a hybridization reaction was performed at room temperature (25 ° C.) for 3 hours. Thereafter, the probe array was added to a 1 M NaCl / 50 mM phosphate buffer (p
H7.0) solution to wash away single-stranded DNA that did not hybridize with the probe nucleic acid. Next, the amount of fluorescence at the spot of the probe array was measured using an inverted fluorescence microscope connected to an image analyzer (trade name: ARGUS 50; manufactured by Hamamatsu Photonics KK) and fitted with a filter set suitable for rhodamine B. Quantified.

(7) Results The spot of the nucleic acid probe of SEQ ID NO: 1, which is a perfect match with the labeled single-stranded DNA, had a fluorescence of 4,600.
Further, after hybridization, the probe array in a state where each spot emits fluorescence was observed using a fluorescence microscope (manufactured by Nikon Corporation). As a result, in the probe array according to the present embodiment, a) each spot is substantially circular and its diameter is in the range of about 70 to 100 μm; b) each spot is located between adjacent spots. It was found that there was a space of about 100 μm, approximately equal to the spot diameter, that each spot was distinctly independent of each other, and c) that the rows and columns of the spots were aligned.

This is extremely effective for automatically detecting hybridized spots on the probe array.

Example 2 (Production of a nucleic acid probe array using a bubble jet printer and detection of a target nucleic acid using the probe array) (1) Exactly the same as (1) and (2) in Example 1 A glass plate having been subjected to a surface treatment for a probe array was prepared.

(2) Synthesis of Probe DNA 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 based on SEQ ID NO: 1 used in Example 1 and have SEQ ID NO: 2 obtained by changing one base and SEQ ID NOs: 3 and 6 obtained by changing 3 bases. The result of the base change was SEQ ID NO: 4.
The single-stranded DNA ends of SEQ ID NOs: 1 to 4 were added to Thiol-Modifier (G
A thiol (SH) group was introduced by using LenResearch. Subsequently, the DNA was recovered by ordinary deprotection, 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- (CH ₂ ) ₆ -O-PO ₂ -O-ACTGGCCGT T GTTTTACA ^3' SEQ ID NO: 3 ^{5 '} HS- (CH ₂ ) ₆ -O-PO ₂ -O-ACTGGCCG CTT TTTTACA ^{3 'SEQ} ID _{^{NO: 4 5' HS- (CH 2}} ) 6 -O-PO 2 -O-ACTGGC ATCTTG TTTACA 3 '(3) binding the SEQ ID NO: 1 to 4 by BJ printer discharge of DNA probes, and the substrate Using the single-stranded DNA, four types of ejection liquids were prepared in the same manner as described in (4) of Example 1 above, and the four inks for the bubble jet printer used in Example 1 were prepared. Tanks were filled with each liquid, and each ink tank was mounted on a bubble jet head. Next, a glass plate prepared in the same manner as in (1) above was mounted on the printer, and 4 g of the glass plate was placed on the glass plate.
Each of the seed nucleic acid probes was placed on a 3 × 3 mm 4 of the glass plate.
Spotted in each of the two areas. The spotting pattern in each area was the same as in Example 1. After the end of the spotting, the glass plate was allowed to stand in a humidified chamber for 30 minutes to react a maleimide group with a thiol group.

(4) Blocking reaction After completion of the reaction between the maleimide group and the thiol group, the glass plate was washed with 1M NaCl / 50 mM phosphate buffer (pH 7.0).
After washing with the solution, the DNA solution on the surface of the glass plate was completely washed away. Next, the glass plate was immersed in a 2% bovine serum albumin aqueous solution and left for 2 hours to perform a blocking reaction.

(5) Hybridization reaction Single-stranded DNA having a base sequence complementary to the DNA of SEQ ID NO: 1 was synthesized by an automatic DNA synthesizer, and rhodamine was bound to the 5 ′ end to label single-stranded DNA. I got 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 a hybridization reaction with the probe array obtained in (4) was performed for 3 hours. . Thereafter, the probe array was prepared using a 1 M NaCl / 50 mM phosphate buffer (pH 7.0).
0) Single-stranded DNA that did not hybridize with the probe nucleic acid by washing with a solution was washed away. Next, each spot of the probe array was observed with a fluorescence microscope (manufactured by Nikon Corporation), and the amount of the fluorescence was measured using an image analyzer (trade name:
ARGUS 50 (manufactured by Hamamatsu Photonics KK) was connected, and quantification was performed using an inverted fluorescence microscope equipped with a filter set suitable for rhodamine B.

(6) Results D of SEQ ID NO: 1, which is a perfect match with the labeled single-stranded DNA
The spot of the NA probe had a fluorescence amount of 4600, whereas the spot of the DNA probe of SEQ ID NO: 2 having a mismatched sequence of 1 base had a fluorescence amount of 2800. In the spot of the DNA probe of SEQ ID NO: 3 having a 3-base mismatch, only a fluorescence amount of 2100 or less than half of the perfect match was obtained, and no fluorescence was observed in the DNA of SEQ ID NO: 4 having a 6-base mismatch. From the above, the single-stranded D which is completely complementary on the DNA array substrate
NA could be specifically detected.

Example 3 (Concentration of DNA Probe in Liquid and Bubble Jet Ejection Characteristics) (1) Synthesis of DNA Probe Single-stranded DNA having the sequence of SEQ ID NO: 5
The solution was synthesized using an NA automatic synthesizer, and the TE solution (10 mM Tris-HCl (pH) was adjusted to a concentration of about 0.2 mg / ml, 2 mg / ml, and 15 mg / ml, respectively.
8) / 1 mM EDTA aqueous solution) to prepare three types of DNA probe solutions having different concentrations (the exact concentration was calculated from the absorption intensity). SEQ ID NO: 5 ^{5 ′} GCCTGATCAGGC ^{3 ′} (2) Discharge by BJ printer 7.5% glycerin, 7.5% urea, 7.5% thiodiglycol, acetylene alcohol having a structure represented by the above general formula (I) (Trade name: acetylenol EH; manufactured by Kawaken Fine Chemical Co., Ltd.) An aqueous solution containing 1% was prepared, and the aqueous solution was adjusted to a concentration of 0.2 m as adjusted in the above (1).
g / ml of probe solution plus a final concentration of about 0.0
Diluted to 2 mg / ml (3 μM). This liquid was filled in the ink tank for the bubble jet printer used in the first embodiment, and this ink tank was mounted on the head of the bubble jet printer used in the first embodiment.

Next, an A4 size aluminum plate was mounted on the printer, and spotting was performed on a 3 × 5 square inch area of the aluminum plate. The spotting here was set so that spotting was performed in the above area at a density of 360 × 720 dpi. First, a commercial ink for BJ620 was printed on the aluminum plate as a control. This operation was performed on a total of four aluminum plates.

Next, the nucleic acid probes spotted on each aluminum plate were recovered using a TE solution, purified by a gel filtration method, and the amount of the purified recovered nucleic acid probes was measured by an absorption spectrum. Here, the recovery amount of the nucleic acid probe theoretically determined is as follows. That is, the volume per droplet ejected from the head of the printer used in this embodiment is 24 picoliter. And 360 × 7
Since there are four aluminum plates spotted in an area of 3 × 5 square inches at a density of 20 dpi, 24 (picoliter) × (720 × 360) × (3 × 5) × 4 sheets = 3
73 μl. 260 n of this amount of probe nucleic acid
absorbance at 300 m and 260 n of the recovered nucleic acid probe
The absorbance at m is shown in FIG.

The same operation as in the above (2) was performed at a density of 2 m
g / ml and 15 mg / ml for each probe solution. The final concentration of the nucleic acid probe in each ejection liquid was 30 μM (0.2 mg / ml) and 225 μM
(1.5 mg / ml). FIG. 3 shows the results of the absorbance of the probe nucleic acid recovered from each solution and the absorbance of the theoretically determined amount of probe nucleic acid.

(3) Results As can be seen from FIG. 3, the actual ejection amount of the nucleic acid probe was close to the theoretically expected value. From this fact, in discharging the nucleic acid probe using the bubble jet method,
No quantitative loss of the nucleic acid probe due to scorching of the nucleic acid probe on the heater portion of the bubble jet head is observed. During the spotting process on the aluminum plate using the liquids of the respective concentrations, no trouble of the head, for example, non-discharge etc. occurred. As a control, spots of ink for a bubble jet printer spotted on an aluminum plate and spots of a nucleic acid probe were visually compared. The spotting conditions of spots formed using liquids having concentrations of 3 μM and 30 μM were almost the same as those of the ink spots. It was similar. In addition, spots formed using a liquid having a concentration of 225 μM were slightly disturbed as compared with ink spots.

Example 4 (Evaluation of Effect of Bubble Jet Process on Nucleic Acid Probe) (1) Synthesis of Nucleic Acid Probe Base length of 10 me consisting of adenine (hereinafter referred to as “A”)
r (synthetic product), oligoA (40-60 mer; manufactured by Pharmacia), poly (dA) (300-400 m
er; Pharmacia Co.) was diluted with a TE solution to prepare a final concentration of 1 mg / ml, and nucleic acid probe solutions having different lengths were prepared. The base sequence of 10 mer (SEQ ID NO: 6) is as follows. SEQ ID NO: 6 ^{5 ′} AAAAAAAAAA ^{3 ′} (2) Discharge of DNA solution by bubble jet printer 7.5% by weight of glycerin, 7.5% by weight of urea and acetylene alcohol represented by the above general formula (I) (trade name:
Acetyleneol EH; Kawaken Fine Chemical) 1 wt%
Is prepared, and each of the nucleic acid probe solutions prepared in the above (1) is prepared using the aqueous solution to a final concentration of about 0.1 mg.
/ Ml.

As in Example 3, each nucleic acid probe solution filled in the cartridge was discharged onto an aluminum plate, and spotting conditions were visually observed. As a result, the base length is 10
For the mer and 40-60 mer nucleic acid probes, a probe array in which independent spots were neatly arranged on an aluminum plate was obtained. In addition, for the 300 to 400 mer nucleic acid probe, basically the same probe array was obtained, but a portion where adjacent spots were connected was recognized. This is considered to be because the physical properties of the liquid were changed due to the long base chain of the nucleic acid probe, and the directionality of ejection from the bubble jet head was slightly inaccurate.

Next, spots on the probe array prepared using the respective nucleic acid probe solutions were collected in the same manner as in Example 3. 100 μl of the collected nucleic acid probe solution was analyzed by reverse phase HPLC, and the presence or absence of cleavage of the nucleic acid probe was examined by comparison with the solution before ejection. The elution by reverse phase HPLC was carried out for 7-70 containing 1 M triethylamine acetate.
Performed with a% acetonitrile gradient. As a result, no DNA fragment considered to have been cleaved was observed, and thus it was confirmed that the nucleic acid probe was not degraded by the ejection by the bubble jet method. Further, as a result of quantifying the recovered nucleic acid probes in the same manner as in Example 3, as shown in FIG. 4, the nucleic acid probes of three different lengths were recovered in almost the same amount as the theoretical value.

Example 5 (Study of Reaction Time) In (4) of Example 1, the surface-treated glass plate on which the nucleic acid probe was spotted was left in a humidified chamber for 10 minutes, 90 minutes, and room temperature (25 ° C.) overnight. A probe array was manufactured in the same manner as in Example 1 except that the probe array was prepared, and each probe array was subjected to a hybridization reaction. As a result, all of the probe arrays reacted for 90 minutes and overnight were given the same fluorescence intensity as that of the probe array obtained in Example 1. From this, it became clear that the binding reaction between the maleimide group on the glass plate surface and the thiol group at the terminal of the nucleic acid probe was almost completed in 30 minutes. On the other hand, the probe array having a reaction time of 10 minutes had a fluorescence amount of about 70% as compared with that of Example 1.

Example 6 (Production of PNA Probe Array Using Bubble Jet Printer and Detection of Target Nucleic Acid Using the Probe Array) A glass plate having been subjected to a surface treatment for a probe array was prepared.

(2) Synthesis of Probe PNA A protein nucleic acid (PNA) (produced by Nippon Perceptive Co., Ltd.) having the base sequences of SEQ ID NOs: 7 and 8 was prepared. This PNA is N-terminal (corresponding to the 5 'end of DNA)
Has a cysteine residue (denoted as Cys), and as a result, a thiol group has been introduced at the N-terminus. The PNA probe of SEQ ID NO: 8 is obtained by changing the PNA probe of SEQ ID NO: 7 by one base. SEQ ID NO: 7 ^N Cys-NH (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -O-CH ₂ CONH-ACTGGCCGTCGTTTTAC
A ^C SEQ ID NO: 8 ^N Cys-NH (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -O-CH ₂ CONH-ACTGGCCGT T GTTTTAC
A ^C (3) Discharge of PNA probe by BJ printer and binding to substrate 100 μl of each of the above PNA probes was 0.1 wt%
Dissolve in trifluoroacetic acid to a final concentration of 80 μM, then 7.5% glycerin, 7.5% urea
%, Thiodiglycol 7.5 wt%, and acetylene alcohol represented by the general formula (I) (trade name: acetylenol EH; manufactured by Kawaken Fine Chemical Co., Ltd.) 1
An aqueous solution containing wt% was added to the PNA trifluoroacetic acid solution to adjust the final concentration of the PNA probe to 8 μM. The surface tension of this liquid is 30-50 dy
n / cm and the viscosity was in the range of 1-5 cps.

Each of the PNA probe solutions was prepared in Example 2.
Spotting was performed on each area on the glass plate prepared in the above (1) in the same manner as described in (3). After the completion of the spotting, the mixture was allowed to stand in a humidified chamber for 3 hours to react a maleimide group with a thiol group.

It should be noted that P per ejection operation of the printer is
The discharge amount of the NA probe solution was about 24 pl.

(4) Blocking reaction After the reaction between the maleimide group and the thiol group was completed, the glass plate was washed with a 1 M NaCl / 50 mM phosphate buffer (pH 7.0).
After washing with the solution, the liquid containing PNA on the surface of the glass plate was completely washed away. Next, the glass plate was immersed in a 2% bovine serum albumin aqueous solution and left for 3 hours to perform a blocking reaction.

(5) Hybridization reaction A single-stranded DNA having a base sequence complementary to PNA of SEQ ID NO: 7 was synthesized by an automatic DNA synthesizer, and a single-stranded DNA was labeled by binding rhodamine to the 5 'end. DNA was obtained. This labeled single-stranded DNA was treated with 10 mM phosphate buffer (pH 7.
0) to a final concentration of 5 nM (solution volume 1 m).
1) The PNA probe array subjected to the blocking treatment obtained in the above (4) was immersed in this DNA solution,
C) for 12 hours.
Thereafter, the probe array was placed in a 10 mM phosphate buffer (pH
7.0) The solution was washed to remove single-stranded DNA that did not hybridize with the PNA probe. Next, the amount of fluorescence at the spot of the probe array was measured using an inverted fluorescence microscope connected to an image analyzer (trade name: ARGUS 50; manufactured by Hamamatsu Photonics KK) and fitted with a filter set suitable for rhodamine B. Quantified.

(6) Results The P of SEQ ID NO: 7, which is a perfect match with the labeled single-stranded DNA
The fluorescence amount of the NA probe was 2,400, whereas that of the PNA probe of SEQ ID NO: 8 having a single base mismatch sequence was 1,100, which is about half. From the above, P
It was possible to specifically detect perfectly complementary single-stranded DNA on the NA array.

After hybridization, the probe array in which each spot emits fluorescence was observed using a fluorescence microscope (manufactured by Nikon Corporation). As a result, in the probe array according to the present embodiment, a) each spot is substantially circular and its diameter is within a range of about 200 μm; b) about 50 μm between adjacent spots There was space, and each spot was clearly independent of each other. C) It became clear that the rows and columns of the spots were aligned.

This is extremely effective in automatically detecting hybridized spots on the probe array.

Further, since it is not necessary to add sodium chloride to the solution used for the hybridization reaction and thereafter for removing unreacted single-stranded DNA, it is not necessary to pay attention to the precipitation of sodium chloride during the observation of fluorescence. Thus, detection of hybrids on the probe array could be performed more easily. Also, there was no need for sealing during storage, and handling was easy.

It is not clear why the spot diameter of the PNA probe is larger than the spot of the probe array obtained in Example 1.
It has been found that the water solubility is slightly inferior to that of the probe. You.

Example 7 (Preparation and Evaluation of Glass Substrate with Black Matrix for Probe Array Having Epoxy Group Introduced on Surface) (1) Glass substrate made of synthetic quartz (50 mm × 50 m
m) was subjected to ultrasonic cleaning using a 2 wt% sodium hydroxide aqueous solution, and then subjected to UV ozone treatment to clean the surface. A 50 wt% methanol aqueous solution containing 1 wt% of a silane coupling agent (trade name: KBM403; manufactured by Shin-Etsu Chemical Co., Ltd.) containing a silane compound (γ-glycidoxypropyltrimethoxysilane) having an epoxy group bonded thereto at room temperature. After stirring for 3 hours, the methoxy group in the silane compound was hydrolyzed. Next, this solution was applied to the surface of the substrate by a spin coater, and heated at 100 ° C. for 5 minutes.
After drying, an epoxy group was introduced on the substrate surface. (2) Next, DEEP-UV containing carbon black
A resist (a negative type resist for a black matrix) (trade name: BK-739P; manufactured by Nippon Steel Chemical Co., Ltd.) is applied by a spin coater so that the film thickness after curing becomes 5 μm.
The substrate was cured by heating at 80 ° C. for 5 minutes on a hot plate. 1cm x 1 using DEEP-UV exposure equipment
In the area of cm, the distance (X) between adjacent wells in FIG.
Is 100 μm, and the shape of the well is 100 μm × 100
Proximity exposure using a mask patterned into a μm square, developed with a developer of an inorganic alkali aqueous solution using a spin developer, and further washed with pure water to completely remove the developer did. Then dry briefly using a spin drier and then dry in a clean oven for 1 hour.
The resist was fully cured by heating at 80 ° C. for 30 minutes to obtain a substrate in which 2500 wells were arranged in a predetermined arrangement and adjacent wells were isolated by a black matrix. The volume of each well is calculated as 50 picoliter (pl). At this time, the contact angle of the black matrix surface to water was 93 °, which was difficult to wet, and the contact angle of the bottom surface of the well to water was 35 °, which was easy to wet.

(3) A 10 μM aqueous solution of Rhodamine B was filled in an ink tank for a bubble jet printer (trade name: BJC620: manufactured by Canon Inc.), and the solution was charged into the bubble jet head of the bubble jet printer used in the first embodiment. I attached it. Then, the solid phase prepared in the above (1) and (2) was mounted on a printer, and a rhodamine B aqueous solution was supplied to the wells of the solid phase in a checkered pattern (every other). The supply amount per well is about 50 pl.
The ejection positioning accuracy of this printer is ± 2.5 μm. Next, an aqueous solution of 10 μM amino FITC was filled in another ink tank, mounted on a bubble jet head of the printer, and supplied to another well adjacent to the well to which the aqueous solution of rhodamine B was previously supplied. Here, rhodamine B and amino FITC were used because they are water-soluble and can be easily ejected from the inkjet head, and the state of liquid supplied to the well and cross contamination can be confirmed by observing fluorescence. is there.

(4) A G excitation filter (for rhodamine B) and a B excitation filter (for amino FITC) were mounted on a fluorescence microscope (manufactured by Nikon Corporation), and each of the components supplied to the well at a magnification of 100 times was used. The state of the aqueous solution was observed with fluorescence. As a result, each aqueous solution was uniformly supplied into the well without forming droplets. Further, no fluorescence of other dyes was observed from each well, and no cross-contamination was observed.

Example 8 (Preparation of Probe Array Using the Substrate of Example 7 and Detection of Target Nucleic Acid Using It) (1) A substrate with a black matrix (BM) was prepared in the same manner as in Example 7. did. (2) an 18-mer oligomer (SEQ ID NO: 9) in which a hydroxyl group at the 5 ′ end is linked to an amino group via a phosphate group and hexamethylene as a DNA probe (SEQ ID NO: 9), one nucleotide for the oligomer of SEQ ID NO: 9 And a probe (SEQ ID NO: 11) having two nucleotides mismatched with the oligomer of SEQ ID NO: 9 (all manufactured by Nippon Flour Milling Co., Ltd., HPLC grade). The base sequence of the oligomer of SEQ ID NO: 9 is M13mp18-ssDNA, which is a single-stranded DNA.
Is a sequence complementary to a part of the nucleotide sequence of the multiple cloning site. The base sequences of SEQ ID NOS: 9 to 11 and the structure of the linkage are shown below. SEQ ID NO: 9 ^{5 '} NH _2- (CH ₂ ) ₆ -O-PO ₂ -O-TGTAAAACGACGGCCAGT ^3' SEQ ID NO: 10 ^{5 '} NH _2- (CH ₂ ) ₆ -O-PO ₂ -O-TGTAAAAC C ACGGCCAGT ^{3 ′} SEQ ID NO: 11 ^{5 ′} NH ₂ — (CH ₂ ) ₆ —O—PO ₂ —O-TGTA T AAC C ACGCCCAGT ^{3 ′} (3) Completely complementary to the above DNA probes of SEQ ID NOs: 9 to 11 Single-stranded DNA was synthesized. Next, each DNA was added to a TE solution (pH 8) containing NaCl at a concentration of 50 mM.
The probe and single-stranded DNA were dissolved to a final concentration of 100 μM, and the DNA probe solution and single-stranded DNA were dissolved.
A solution was prepared. And a solution 10 containing a DNA probe.
To 0 μl, 100 μl of a solution containing single-stranded DNA complementary to each DNA probe was added and mixed, and each mixed solution was cooled linearly from 90 ° C. to 25 ° C. over 2 hours. A hybrid of the probe and each single-stranded nucleic acid was formed. Next, the above SEQ ID NOs: 9 to 11
A solution containing a hybrid of each of the DNA probes described above was prepared by mixing 7.5 wt% of glycerin, 7.5 wt% of urea, 7.5 wt% of thiodiglycol, and acetylene alcohol represented by the general formula (I) (trade name: acetylenol EH; It was added to an aqueous solution containing 1 wt% (Kawaken Fine Chemical Co., Ltd.), and the final concentration of the hybrid was adjusted to 8 μM. The surface tension of each of these liquids containing the hybrid of each DNA probe is 30 to 50 dyne /
cm and the viscosity was within a range of 1 to 5 cps (E-type viscometer: manufactured by Tokyo Keiki Co., Ltd.).

Next, a bubble jet printer (trade name:
Three ink tanks for BJC620 (manufactured by Canon Inc.) were prepared, and each of the ink tanks was filled with the above-mentioned three types of hybrid solutions, and was attached to the head of the bubble jet printer used in Example 1. Further, the glass substrate with BM prepared in the above (1) and (2) was set, and first, a solution containing the hybrid of the DNA probe of SEQ ID NO: 9 was supplied to the well in the first row (131 in FIG. 6). Next, a solution containing the hybrid of the DNA probe of SEQ ID NO: 10 is supplied to the second row day well (133 in FIG. 6) adjacent to the well of the first row, and the solution containing the hybrid of the DNA probe of SEQ ID NO: 11 is further supplied. Was supplied to the wells in the third row (135 in FIG. 6) adjacent to the wells in the second row. Each hybrid solution was discharged four times to one well and supplied at about 100 pl. Although this volume is about twice the volume of one well, when each well was observed with a microscope, the supplied hybrid solution was raised from the opening of the well. It stayed in the well and no cross-contamination between the wells was observed.

Next, the substrate was placed in a thermo-hygrostat at 25 ° C. and 100% humidity for 12 hours to allow the amino group of the probe to react with the epoxy group of the well. In addition, since the amino group of the base of the probe forms a hybrid with the completely complementary single-stranded DNA, it does not react with the epoxy group in each well.

(4) Next, the substrate was washed with pure water at 80 ° C. for 10 minutes to dissociate and wash away the complementary strand forming a hybrid with the probe bound to the substrate. Next, the substrate was treated with a 1% aqueous solution of ethanolamine for 1 hour at room temperature to open unreacted epoxy groups in each well. Next, the substrate was washed with pure water and dried.

The DN in the well is obtained by repeating the above (4).
The epoxy group that did not react with the A probe is opened to form a hydroxyl group, and the hydroxyl group is also present in the reacted ethanolamine, so that the bottom surface of the well becomes more hydrophilic,
This is advantageous when a solution containing the target single-stranded DNA described below is supplied to the well.

(5) Next, a single-stranded DNA that is completely complementary to the DNA probe of SEQ ID NO: 9 was added to a TE solution (pH 8) containing NaCl at a concentration of 50 mM at a final concentration of 10 μM.
The probe array having the epoxy group introduced into the well obtained in the above (4) was immersed in this solution,
The temperature was lowered from 0 ° C. to 25 ° C. over 2 hours to carry out a hybridization reaction. Then, at 20 ° C., 10 mM N
After washing the substrate with a TE buffer solution (pH 8) containing aCl for 20 minutes, the washing solution on the surface was removed with a spin dryer.

(6) Next, 2-methyl-4,6-methyl which emits fluorescence only when intercalated into a double-stranded nucleic acid is added to a TE solution (pH 8.0) containing NaCl at a concentration of 50 mM.
Bis (4-N, N-dimethylaminophenyl) pyrylium iodide (hereinafter abbreviated as "P2") having a concentration of 10
The solution was dissolved to a concentration of μM, and the solution was filled in the ink tank for the ink jet printer and attached to the head of the ink jet printer. Further, the substrate subjected to the hybridization in the above (5) is set in the printer, and the P2 solution is added to each well with 100 μl.
pl after each supply, humidity 10 to prevent drying
It was left in a dedicated chamber of 0% for 5 minutes, and an inverted microscope (trade name: IMT2; manufactured by Olympus Optical Co., Ltd., magnification: 100 times, filter cube for fluorescence microscope (excitation filter 455 nm)
From 595 nm (transmission), dichroic mirror 620
nm, fluorescence barrier filter from 610 nm to 725
nm (transmission))) for the ICCD camera (trade name: C
2400-87; Hamamatsu Photonics, Inc.) and an image processor (trade name: ARGUS 50; Hamamatsu Photonics, Inc.) were connected, and the fluorescence was observed and quantified. The observation area was 25 μm × 25 μm, integration × 64, AR
The amplification level of GUS50 was set appropriately.

As a result, from the well to which the DNA probe of SEQ ID NO: 11 was bound, fluorescence intensities of 1200 to 1500, almost similar to the background, were observed. On the other hand, the fluorescence intensity of 9800 to 10300 was observed from the well to which the DNA probe of SEQ ID NO: 9 was bound, and the fluorescence intensity of 3500 to 3900 was observed from the well to which the DNA probe of SEQ ID NO: 10 was bound. . Further, each solid phase was washed with a TE buffer at 35 ° C. for 10 minutes, and the fluorescence intensity was measured again.
Only the same fluorescence intensity as the background was observed from the wells to which the probe was bound.

From these results, it is possible to carry out a hybridization reaction in each well by using the probe array according to the present example.
It was found that the target nucleic acid completely complementary to the target nucleic acid could be specifically detected.

Example 9 (Selective supply of reactants to each well of probe array of Example 8 and reaction with probe) (1) DNAs of SEQ ID NOS: 9 to 11 in the same manner as in Example 8
A substrate to which the probe was bound was prepared.

(2) Three types of single-stranded DNAs completely complementary to the DNA probes of SEQ ID NOS: 9 to 11 were synthesized. TE solution containing NaCl at a concentration of 50 mM (pH
8) The above three types of single-stranded DNA were added to each other at a concentration of 100.
It dissolved so that it might be set to μM. Three ink tanks for a bubble jet printer (trade name: BJC620; manufactured by Canon Inc.) are prepared, and the above three ink tanks are provided in each ink tank.
The seeded single-stranded DNA solution was filled and mounted on the head of the bubble jet printer used in Example 1. The substrate prepared in the above (1) was also set on the printer, and a solution containing single-stranded DNAs completely complementary to the wells to which the DNA probes of SEQ ID NOS: 9 to 11 were bound was added in an amount of 100 pl per well. Supplied one by one. At this time, when the state of each well was observed with a microscope, it was found that bleeding of the solution and no cross-contamination were observed, and that a solution of a substance to be reacted individually could be supplied to each well of the probe array.

(3) Next, after performing a hybridization reaction in each well in the same manner as in Example 8,
In the same manner as in Example 8, the P2 solution was supplied to each well, and the hybrid was detected by observing the fluorescence. As a result, fluorescence having an intensity of 9800 to 10300 was observed from all the wells. From this, it was confirmed that a reactant was individually supplied to each well of the solid-phase probe array, the probe was reacted with the reactant in each well, and a reaction product could be detected.

Example 10 (Hydrophilic treatment of bottom surface of well of substrate of Example 7) (1) A glass substrate having a black matrix pattern was prepared in the same manner as in Example 7.

(2) UV ozone treatment was performed on the surface of the substrate on which the black matrix was formed.
At this time, the contact angle of water on the black matrix surface was 93 °, which is hard to wet, and the contact angle of water on the bottom surface of the well was 22 °, which was compared with that of the well bottom surface of the substrate with black matrix obtained in Example 7. It was in a state that was easy to get wet. This is considered to be the effect of the UV ozone treatment.

(3) Next, the supply state of the ink-jet discharge liquid to the wells was observed using an aqueous solution of rhodamine B and amino FITC in the same manner as in Example 7, and each aqueous solution was dropped in the well. Was uniformly supplied into the wells without forming the same. When a solid phase with a well on the surface is used as the solid phase of the probe array, the inkjet discharge liquid is limited as much as possible, unlike a solid phase with no flat surface and uniform surface characteristics. It is not necessary to keep the ink at the set position, and rather, it is more advantageous to sufficiently spread the ink-jet ejection liquid on the bottom of the well for detecting a reaction between the probe and the target substance performed later. The hydrophilization treatment of the well bottom surface described in this example is a preferable method as one embodiment thereof. In addition, no other dye was observed from the well to which each dye was supplied, and it was found that each dye aqueous solution could be supplied to each well using the inkjet process without causing cross contamination. Was.

Example 11 (A method for producing a probe array using a solid phase obtained by supplying a liquid for introducing a functional group for immobilizing a probe into each well of a BM forming substrate by an ink-jet method and its use) (1) A substrate provided with a black matrix was prepared in the same manner as in Example 7.

(2) A silane coupling agent containing an amino-bonded silane compound (N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane) (trade name: KBM603; manufactured by Shin-Etsu Chemical Co., Ltd.) 1wt
% Of a 10 wt% methanol aqueous solution was stirred at room temperature for 3 hours to hydrolyze the methoxy group in the silane compound. Next, this solution was filled in an ink tank for a bubble jet printer (trade name: BJC620; manufactured by Canon Inc.), and attached to the head of the bubble jet printer used in Example 1. The substrate prepared in the above (1) was also set in the printer, and a silane coupling agent solution containing a silane compound having a methoxy group hydrolyzed was supplied to the well in the same manner as in Example 8. 25
After leaving for 30 minutes in a thermo-hygrostat at 100 ° C and 100% humidity,
The substrate was washed with pure water, spin-dried, and baked at 100 ° C. for 30 minutes to introduce an amino group into the bottom surface of each well.

(3) Next, succiimidyl-4- (maleimidophenyl) butyrate (manufactured by Aldrich) was added to 5 w
The solution was dissolved in a t% DMSO solution so as to have a final concentration of 5 wt%, and this solution was supplied to each well by an ink jet printer in the same manner as in (2) above, 100 pl at a time. The substrate was left in the bath for 2 hours. Next, the substrate was washed pure and spin-dried to introduce a maleimide group on the bottom of each well.

(4) An 18-mer oligomer (SEQ ID NO: 12) in which a hydroxyl group at the 5 'end is linked to a thiol group via a phosphate group and hexamethylene as a DNA probe (SEQ ID NO: 12) A probe whose nucleotides are mismatched (SEQ ID NO: 13) and a probe whose nucleotides are mismatched with respect to the oligomer of SEQ ID NO: 12 (SEQ ID NO: 14) (all manufactured by Nippon Milling Co., Ltd., HPLC grade) are prepared. did. The base sequences of SEQ ID NOS: 12 to 14 and the structure of the linkage are shown below. SEQ ID NO: 12 ^{5 '} HS- (CH ₂ ) ₆ -O-PO ₂ -O-TGTAAAACGACGGCCAGT ^3' SEQ ID NO: 13 ^{5 '} HS- (CH ₂ ) ₆ -O-PO ₂ -O-TGTAAAAC C ACGGCCAGT ^3' SEQ ID NO: 14 ^{5 '} HS- (CH ₂ ) ₆ -O-PO ₂ -O-TGTA T AACCACG C CCAGT ^3' (5) The above SEQ ID NOS: 12 to 1 in a 10 mM phosphate buffer.
Each DNA probe of No. 4 was dissolved to a final concentration of 10 μM. Each DNA probe solution was supplied to the well of the substrate prepared in (3) in the same manner as in Example 8 above. Observation of each well with a microscope revealed that the supplied DNA probe solution was raised from the well opening, but was stopped in the well by the hydrophobic matrix, and no cross-contamination was observed. The substrate was released in a thermo-hygrostat at 30 ° C. and 100% humidity for 2 hours, and then washed with pure water and spin-dried to react the thiol group of each DNA probe with the maleimide group of each well. Was bonded to the substrate.

(6) A single-stranded DNA completely complementary to the DNA probe of SEQ ID NO: 12 was synthesized,
This single-stranded DNA was dissolved in a TE solution containing 0 mM in a concentration of 10 μM. The DNA probe-bound substrate obtained in the above (5) is immersed in this solution,
The temperature was lowered from 2 to 25 ° C over 2 hours to perform hybridization. Next, the substrate was washed at 20 ° C. for 20 minutes using a TE solution (pH 8) containing NaCl at a concentration of 10 mM, and then the cleaning solution on the substrate surface was removed with a spin dryer.

(7) YOYO-1 which is a reagent that emits fluorescence only after intercalation into a hybrid is replaced with Na
A final concentration of 10 was added to a TE solution containing Cl at a concentration of 50 mM.
The solution was dissolved to a concentration of μM (pH 8). This solution was added to each well of the substrate which had been subjected to the treatment of the above (6) using an ink jet printer in the same manner as in the above (2).
The fluorescence was observed and quantified in the same manner as in Example 8 (using a B excitation filter). The signal amplification level of Argus 50 is the same as that of the eighth embodiment.

As a result, from the well to which the DNA probe of SEQ ID NO: 14 was bound, a fluorescence intensity of 1800 to 2000, almost the same as the background, was observed. On the other hand, fluorescence intensity of 7500 to 8000 was observed from the well to which the DNA probe of SEQ ID NO: 12 was bound, and fluorescence intensity of 3100 to 3300 was observed from the well to which the DNA probe of SEQ ID NO: 13 was bound. . Further, the solid phase was washed with a TE buffer at 35 ° C. for 10 minutes, and the fluorescence intensity was measured again. From the well to which the DNA probe of SEQ ID NO: 13 was bound, only the fluorescence intensity equivalent to the background was observed. lost.

From these results, the hybridization reaction can be carried out in each well by using the probe array according to the present example.
It was found that the target nucleic acid completely complementary to the target nucleic acid could be specifically detected.

Example 12 (1) In the same manner as in Example 11, D of SEQ ID NOS: 12 to 14
A substrate to which an NA probe was bound was prepared.

(2) Three types of single-stranded DNAs completely complementary to the DNA probes of SEQ ID NOS: 12 to 14 were synthesized. The above 3 was added to a TE solution containing 50 mM NaCl.
Each type of single-stranded DNA was dissolved so that each concentration was 10 μM. The pH of each single-stranded DNA solution is 8. Bubble jet printer (product name: BJC62)
0; manufactured by Canon Inc.), three ink tanks were prepared, and each of the ink tanks was filled with the above-mentioned three types of single-stranded DNA solutions, and attached to the head of the bubble jet printer used in Example 1. did. The substrate prepared in the above (1) was also set in the printer, and a solution containing single-stranded DNAs completely complementary to the wells to which the DNA probes of SEQ ID NOs: 12 to 14 were bound was added in an amount of 100 pl per well.
Supplied one by one. At this time, when the state of each well was observed with a microscope, it was found that bleeding of the solution and no cross-contamination were observed, and that a solution of a substance to be reacted individually could be supplied to each well of the probe array.

(3) Next, after performing a hybridization reaction in each well in the same manner as in Example 11, the YOYO-1 solution was supplied to each well in the same manner as in Example 11, and the fluorescence was observed. The detection of hybrid was carried out. As a result, 7500 to 8
A fluorescence intensity of 000 was observed. From this, it was confirmed that a reactant was individually supplied to each well of the solid-phase probe array, the probe was reacted with the reactant in each well, and a reaction product could be detected.

Example 13 (Production method of probe array using substrate in which BM-formed substrate was immersed in a solution for introducing an epoxy group and epoxy group was introduced into a well) (1) As described in (2) of Example 7 A substrate with a black matrix was prepared.

(2) As described in (1) of Example 7,
A 1 wt% aqueous solution of a silane coupling agent (trade name: KBM403; manufactured by Shin-Etsu Chemical Co., Ltd.) containing a silane compound (γ-glycidoxypropyltrimethoxysilane) having an epoxy group bonded thereto is stirred at room temperature for 1 hour. The methoxy group in the molecule of the silane compound was hydrolyzed. Next, the solid phase prepared in the above (1) is added to this solution at room temperature for 3 hours.
After immersion for 0 minutes, the solid phase was washed with pure water, water was removed with a stream of nitrogen gas, and baked at 120 ° C. for 5 minutes to introduce an epoxy group into the bottom of the well. At this time, the contact angle of the BM surface to water was 95 °, which was hard to wet, and the contact angle of the bottom of the well to water was 33 °, which was easy to wet. Thus, the epoxy group can be introduced to the bottom of the well by treating the solid phase after the formation of the BM with the silane coupling agent.

(3) The DNA probes of SEQ ID NOS: 9 to 11 were bound to the bottom of the well according to the method described in (3) and (4) of Example 8 above.

(4) A single-stranded DNA having a base sequence complementary to SEQ ID NO: 9 is synthesized by an automatic DNA synthesizer,
A labeled single-stranded DNA was obtained in which tetramethylrhodamine was bound to the 5 'end via a hexanolamine linker. The labeled single-stranded DNA was dissolved in a TE solution (pH 8) containing NaCl at a concentration of 50 mM to a final concentration of 2 μM. The DNA probe-bound substrate obtained in the above (3) was immersed in this solution, and the temperature was lowered from 80 ° C. to 25 ° C. over 2 hours to perform a hybridization reaction. Thereafter, the probe array was replaced with 10 mM NaCl / TE.
The single-stranded DNA that did not hybridize with the probe nucleic acid was washed away at 29 ° C. for 20 minutes using a buffer solution (pH 8). Next, the amount of fluorescence from each well was quantified in the same manner as in Example 8.

(5) Results D of SEQ ID NO: 9 which is a perfect match with the labeled single-stranded DNA
8500 to 94 from the well to which the NA probe was bound
A fluorescence amount of 00 was confirmed. DNA of SEQ ID NO: 10
2800-3400 from the well with the probe attached
Was observed, and only about 1200 to 1500 fluorescence was observed from the well to which the DNA probe of SEQ ID NO: 11 was bound. When the above probe array was further washed with a 10 mM NaCl / TE buffer (pH 8) at 35 ° C. for 10 minutes, the DN of SEQ ID NO: 10 was obtained.
The amount of fluorescence from the wells with the A probe bound to background levels. Therefore, it can be seen that specific detection of a hybrid target substance is possible even using the probe array according to the embodiment.

[0155]

As described above, according to the present invention, the spot containing the probe can be formed on the solid phase by using the ink-jet technique without damaging the probe and without generating a satellite spot. Can be spotted. Also, by using this method, a high-quality probe array having probe spots independently and at a high density can be efficiently manufactured.

Further, according to the present invention, a probe array capable of more accurately examining more information on a target substance even from a small amount of a specimen can be obtained. It can be more accurately and quickly determined whether or not to perform. Similarly, by using this probe array, the structure of the target substance in the sample can be specified more accurately and quickly.

Further, according to the present invention, a matrix pattern is formed on the surface as a solid phase of a probe array, and a solid phase provided with a well is used to supply a probe solution to the solid phase or a sample to the solid phase. It is possible to cope with a slight misalignment of the supply. Further, by supporting various functions on the matrix, it has become possible to further improve the accuracy of detection of a target substance, identification of a structure, and the like.

[0158]

[Sequence list] SEQ ID NO: 1 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Other information: 5'-terminal thiol group Sequence: ACTGGCCGTCGTTTTACA SEQ ID NO: 2 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Other information: Thiol group at 5 'end Is a binding sequence ACTGGCCGT T GTTTTACA SEQ ID NO: 3 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ACTGGCCG CTT TTTTACA SEQ ID NO: 4 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ACTGGC ATCTTG TTTACA SEQ ID NO: 5 Sequence length: 12 Sequence type: Nucleic acid Number of strands: single strand Topology: linear Sequence type: Other nucleic acid Synthetic DNA sequence GCCTGATCAGGC SEQ ID NO: 6 Sequence length: 10 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AAAAAAAAAA sequence Number: 7 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic PNA Other information: Cysteine residue bonded to N'-terminal Sequence ACTGGCCGTCGTTTTACA SEQ ID NO: 8 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic PNA Other information: Cysteine residue bonded to N 'end Sequence ACTGGCCGT T GTTTTACA SEQ ID NO: 9 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Other information: Amino group at 5 'end Binding sequence TGTAAAACGACGGCCAGT SEQ ID NO: 0 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Other information: Amino group bonded to 5 'end Sequence TGTAAAACCACGGCCAGT SEQ ID NO: 11 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Other information: 5'-terminal amino group bonded Sequence TGTATAACCACGCCCAGT SEQ ID NO: 12 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Other information: 5'-terminal bonded thiol group Sequence TGTAAAACGACGGCCAGT SEQ ID NO: 13 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Other information: 5'-terminal thiol group attached Sequence TGTAAAACCACGGCCAGT SEQ ID NO: 14 Length of sequence: 1 8 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Other information: A thiol group is bonded to the 5 'end Sequence TGTATAACCACGCCCAGT

[Brief description of the drawings]

FIG. 1 is a schematic explanatory view of a method for manufacturing a probe array using a bubble jet head.

FIG. 2 is a sectional view taken along line AA of the bubble jet head of FIG.

FIG. 3 is a graph comparing the theoretical value of the amount of nucleic acid probe spotted on an aluminum plate by the bubble jet method in Example 3 with the actual recovered amount.

FIG. 4 is a graph comparing the theoretical value of the amount of nucleic acid probe spotted on an aluminum plate by the bubble jet method in Example 4 with the actual recovered amount.

FIG. 5 (a) is a schematic plan view of one embodiment of a probe array according to the present invention. FIG. 6B is a sectional view taken along line BB of FIG.

FIG. 6 is an explanatory diagram of a spotting method according to an eighth embodiment.

[Explanation of symbols]

Reference Signs List 101 Nozzle 103 Solid phase 104 Droplet 105 Bubble jet head 107 Discharged liquid containing nucleic acid probe 109 Protective film 111-1, 111-2 Electrode 113 Heating resistor layer 115 Heat storage layer 116 Formed with alumina having good heat dissipation Substrate 117 Heating Head 119 Discharge Orifice 121 Meniscus 123 Bubble Area

Claims (10)

[Claims] 1. A liquid composition for discharging a probe capable of specifically binding to a target substance by an ink jet method, comprising a probe, urea, glycerin, thiodiglycol and the following general formula (I). A liquid composition comprising: (In the above formula, R ₁ , R ₂ , R ₃ and R ₄ represent an alkyl group, m and n each represent an integer, and m = 0 and n =
0 or 1 ≦ m + n ≦ 30, and when m + n = 1, m or n is 0. ) 2. Urea is added to the liquid composition in an amount of 5 to 10 w.
2. The liquid according to claim 1, wherein the liquid contains t%, glycerin in an amount of 5 to 10% by weight, thiodiglycol in an amount of 5 to 10% by weight, and the acetylene alcohol represented by the general formula (I) in an amount of 0.02 to 5% by weight. Composition. 3. The method according to claim 1, wherein the concentration of the probe in the liquid composition is 0.5.
The liquid composition according to claim 1, wherein the liquid composition has a concentration of from 0.5 to 500 μM. 4. The method according to claim 1, wherein the concentration of the probe in the liquid composition is 2 to 4.
The liquid composition according to claim 3, which is 50 µM. 5. The method according to claim 1, wherein the probe is a single-stranded nucleic acid.
5. The liquid composition according to any one of items 1 to 4, 6. The single-stranded nucleic acid has a length of 2 to 5000 mer.
The liquid composition according to claim 5, which is: 7. The liquid composition according to claim 6, wherein the length of the single-stranded nucleic acid is 2 to 60 mer. 8. The liquid composition according to claim 1, wherein the single-stranded probe is a single-stranded DNA. 9. The liquid composition according to claim 1, wherein the probe is a single-stranded RNA. 10. The liquid composition according to claim 1, wherein the probe is a single-stranded PNA.