WO2007069608A1

WO2007069608A1 - Process for production of probe-immobilizing supports, process for production of dna arrays, and probe-immobilizing supports

Info

Publication number: WO2007069608A1
Application number: PCT/JP2006/324770
Authority: WO
Inventors: Keisuke Makino; Mitsuru Nonogawa; Yasuko Yoshida; Kazunari Yamada
Original assignee: Ngk Insulators, Ltd.
Priority date: 2005-12-13
Filing date: 2006-12-12
Publication date: 2007-06-21
Also published as: JPWO2007069608A1

Abstract

A process for the production of probe-immobilizing supports which comprises the step (a) of reacting a solid support having hydroxyl groups on the surface with one or more linker organosilane compounds having linker sites capable of binding to a probe to bond the silicon atoms of the linker organosilane compounds to oxygen atoms of the hydroxyl groups and thereby make the organosilane compounds held on the solid support and the step (b) of protecting the residual hydroxyl groups on the surface of the solid substrate with a capping agent.

Description

Specification

Method for producing probe immobilization carrier, DNA array production method, and probe immobilization carrier

Technical field

The present invention relates to a method for producing a probe-immobilizing carrier, a method for producing a DNA array, and a probe-immobilizing carrier.

Background art

Conventionally, various techniques relating to DNA arrays have been developed. For example, Non-Patent Document 1 describes a method of immobilizing a DNA probe on the surface of a solid phase substrate. In this Non-Patent Document 1, in order to immobilize a DNA probe on the surface of a solid phase substrate, the surface treatment of the solid phase substrate such as glass or silica is performed, and as the surface treatment agent, an amino group, an aldehyde group, The use of various silane coupling agents having an epoxy group or the like is described.

Non-Patent Document 1: Fumio Kimizuka, Yasuyuki Kato, “Protein Nucleic Acid Enzyme” Vol. 43, No. 13 (1988), p52- 59

Disclosure of the invention

However, even if the surface treatment of the solid phase substrate is performed with various silane coupling agents, not all hydroxyl groups present on the surface of the solid phase substrate are bonded to the silane coupling agent. Since some of the hydroxyl groups remain, some problems arise due to the remaining hydroxyl groups. For example, since the hydroxyl group remaining on the solid phase substrate becomes a strong acid in the presence of moisture, the strong acid may cut between the silane coupling agent and the solid phase substrate after long-term storage. In this case, there is a problem that sufficient fluorescence intensity cannot be obtained even if hybridization is performed between a DNA probe and a target labeled with a fluorescent substance. The remaining hydroxyl group is considered to reduce the reactivity by chemically bonding with the amino group of the silane coupling agent. If the reactivity of the amino group, etc. of the silane coupling agent decreases, a sufficient amount of DNA probe cannot be held on the solid phase substrate, so that the hybrid of the DNA probe and the target labeled with a fluorescent substance is still present. Seesho Even if the process is performed, there is a problem that sufficient fluorescence intensity cannot be obtained. In this specification, such a silane coupling agent is referred to as a linker, and an amino group or the like that the silane coupling agent has is referred to as a linker partial position.

[0004] One object of the carrier for immobilizing a probe and the method for producing the same of the present invention is to stably hold a linker that binds to a probe such as a nucleic acid on a solid phase substrate. Another object is to enable the linker on the solid phase substrate to be efficiently bonded to the probe.

The present invention employs the following means in order to solve at least one of the above-mentioned objects.

[0006] The first method for producing a probe-immobilizing carrier of the present invention comprises:

(a) reacting a solid phase substrate having a hydroxyl group on the surface with one or more types of organosilane compounds for linkers having a partial linker position capable of binding to a probe, and using the linker for oxygen atoms of the hydroxyl groups A step of holding the organosilane compound for a linker on the surface of the solid phase substrate as a linker by bonding a silicon atom of the organosilane compound;

(b) a step of protecting a hydroxyl group remaining on the surface of the solid phase substrate after the step (a) with a caving agent;

Is included.

[0007] According to this method for producing a probe-immobilizing carrier, the possibility that a hydroxyl group remains on the surface of the solid phase substrate is reduced, so that the hydroxyl group on the solid phase substrate is bonded to the linker by the remaining hydroxyl group. It is less likely that the linker is cleaved or the linker part held on the solid phase substrate does not easily react with the probe. Therefore, when the obtained probe immobilization carrier and the probe are reacted, a sufficient amount of the probe can be held on the probe immobilization carrier. Here, in the step (b), one or two or more kinds of organosilane compounds for cabbing not having the partial linker position are used as the cabbing agent, and the solid phase substrate after the step (a) And bonding the oxygen atom of the hydroxyl group remaining on the surface of the organic silane compound for the cabbing to the oxygen atom of the hydroxyl group.

An example of this production method scheme is shown in FIG. An example of the probe-fixing carrier obtained by this manufacturing method is shown in FIGS. 2 (a) and 2 (b). Cap agent is cap To protect the hydroxyl groups on the solid phase substrate. The cap may be a monomer cap shown in FIG. 3 or a polymer cap shown in FIG. Further, as shown in Fig. 5, a plurality of types of linkers may be mixed.

[0009] The second method for producing a probe-immobilizing carrier of the present invention comprises:

(a) One or two or more types of organosilane compounds for a linker having a partial linker position capable of binding to a probe with respect to a solid phase substrate having a hydroxyl group on the surface, the oxygen atom of the hydroxyl group and the linker Introducing the organic silane compound through a bond with a silicon atom;

(b) Functionality such that the interaction of the linker with the partial position of the linker and the Z or electrostatic interaction with the solid phase substrate after the step (a) is smaller than the hydroxyl group. Introducing one or two or more types of organosilane compounds for cabbing having a group through a bond between an oxygen atom of the hydroxyl group and a key atom of the organosilane compound for a linker;

Is included.

[0010] According to this method for producing a probe-fixing carrier, the possibility that a hydroxyl group remains on the surface of the solid phase substrate is reduced, so that the hydroxyl group on the solid phase substrate binds to the linker by the remaining hydroxyl group. Are less likely to be cleaved or the linker part held on the solid phase substrate is less likely to react with the probe. Therefore, when the obtained probe immobilization carrier and the probe are reacted, a sufficient amount of the probe can be held on the probe immobilization carrier.

[0011] In the method for producing a probe-immobilizing carrier carrier of the present invention, the organic silane compound for cabling may be a compound having an alkoxy group or a halogen group in addition to an alkyl group on a key atom. In this way, the alkoxy group or halogen group of the organosilane compound for caving is eliminated, and the silicon atom of the silane compound and the oxygen atom of the solid phase substrate are easily bonded. Such an organosilane compound for caving is not particularly limited, but may be, for example, one or more compounds represented by the formula (1). When X is an alkoxy group, the alkoxy group is one or two selected from a methoxy group, an ethoxy group, and an n-propoxy group (preferably a methoxy group and an ethoxy group). It may be the above. R is a hydrocarbon group, more preferably a straight chain, which is preferably a straight chain or branched alkyl group having 1 to 20 (preferably 1 to 8) carbon atoms. Examples of such hydrocarbon groups include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, and an n-octadecyl group. More specifically, as one or two or more compounds selected from the group consisting of formulas (2) to (8).

[Chemical 1]

SiX R

m n

(In the formula (1), m and n are each an integer of 1 or more, m + n is 4, X is a halogen atom such as bromine, chlorine, iodine, or an alkoxy group, and m is 2 or more. Sometimes X may be the same or different. R is a hydrocarbon group, and when n is 2 or more, R may be the same or different.)

[Chemical 2]

BMS

MMS

ODTES

■ (8)

A ₀

ETMS In the method for producing a probe-immobilizing carrier carrier of the present invention, the organosilane compound for a linker may have an alkoxy group and a group having a terminal partial position of the linker on a silicon atom. Alternatively, the organosilane compound for a linker is one or more selected from the group consisting of an amino group, a thiol group, an ester group, an aldehyde group, an epoxy group, and a carboxyl group as the linker partial position. It may have a group. The solid phase substrate may be made of glass, plastic or silicon. In addition, it has a hydroxyl group on the surface of the solid phase substrate! / Cunning! In the case of drought, hydroxyl groups are generated on the surface by performing treatment according to the material of the solid phase substrate. Here, an example of a linker is shown in FIG. Figure 6 shows a linker with a monomeric organosilane moiety. Two types of linkers are bonded to oxygen atoms on the solid phase substrate, but only one type of linker may be bonded. More than one type of linker may be combined. The linker may be a linear hydrocarbon chain having 1 to 10 carbon atoms having a terminal partial linker position. Figure 7 shows another example of a linker. FIG. 7 shows a linker having an organosiloxane moiety (or an organic polysiloxane moiety).

[0014] In the method for producing a DNA array of the present invention, a probe is introduced into the probe solidifying carrier produced by any of the above-described methods for producing a probe immobilizing carrier using the partial position of the linker. To obtain a DNA array. In this way, a DNA array in which a sufficient amount of probes are retained can be obtained, so that sufficient fluorescence intensity is obtained, for example, when hybridization between a probe and a target compound labeled with a fluorescent substance is performed. can get.

Here, an example of a DNA array is shown in FIGS. 8 (a) and 8 (b). Fig. 8 (a) is an example when a linker having an organic siloxane moiety or an organic polysiloxane moiety is used, and Fig. 8 (b) is a monomer and a linker having an organosiloxane moiety or an organic polysiloxane moiety. This is an example in the case of using a mixture of linkers having an organosilane moiety.

In the method for detecting a target compound of the present invention, the target compound is detected by the probe by supplying a test sample containing the target compound that interacts with the probe to the DNA array described above. Alternatively, the method includes a step of detecting the signal that is changed or generated by the interaction between the probe and the target compound by capturing the target compound with the probe. In this way, a target compound or signal can be detected with high sensitivity because a DNA array holding a sufficient amount of probe is used.

[0017] The first probe-immobilizing carrier of the present invention is produced by any one of the above-described methods for producing a probe fixing rod carrier. The second probe-immobilizing carrier of the present invention has a solid-phase substrate and a linker atom that can bind to the probe, by binding of a key atom to an oxygen atom on the surface of the solid-phase substrate. One or two or more types of linkers held on the surface of the solid phase substrate and oxygen atoms on the surface of the solid phase substrate And a cap that protects the one to which the linker is not bonded. The third probe-immobilizing carrier of the present invention comprises a solid phase substrate, one or more linkers having a linker partial position that is held on the surface of the solid phase substrate and can bind to the probe, and a hydrocarbon. One or two or more kinds of caps that have a system functional group and are held on the surface of the solid-phase substrate through an oxygen atom and a key atom bond. A sufficient amount of the probe can be retained by reacting with the probe by any of the first to third carriers for probe immobilization according to the present invention. Here, the cap has a hydrogen bond interaction or electrostatic interaction with the linker partial position of the linker that is based on a hydrogen bond interaction or electrostatic interaction between the linker site and a hydroxyl group. It is preferable to have a small functional group. In this way, it is possible to effectively prevent the remaining hydroxyl group from chemically bonding with the partial position of the linker and hindering the reaction with the probe.

[0018] In the present invention, the organosilane compound for a linker and the organosilane compound for a cabbage are each one or more and three or less hydrolyzable groups X (for example, no, rogen atom or An alkoxy group, etc.). In addition, these organosilane compounds each have one hydrolyzable group X, two hydrolyzable groups X, and three hydrolyzable groups X. It may be configured, but these hydrolyzable groups X may be combined in two or more different types. Further, an organosilane compound having one hydrolyzable group X and an organosilane compound having two or more hydrolyzable groups may be combined. For example, an organic silane compound having one hydrolyzable group X and an organic silane compound having the same three may be used in combination, or an organic silane compound having the same and two organic silane compounds. Or two organic silane compounds and three organic silane compounds, or a combination of the same, two, and three organic silane compounds. That's right.

[0019] As an invention related to the present invention, the following configuration may be adopted.

[0020] (1) In the above-described method for producing a probe-immobilizing carrier carrier according to the present invention, the step (a) includes the partial linker position as the organosilane compound for a linker, A first linker-containing organosilane compound having a hydrolyzable group X, and a partial position of the linker; In addition, a second organosilane compound for a linker having two or more hydrolyzable groups X may be used. An example of the first and second organosilane compounds for the linker used in this production method is shown in FIG.

[0021] (2) In the manufacturing method according to (1), in the step (a), the second organic organosilane compound for the linker is used together with the first organic organosilane compound for the linker. It is good also as the process of supplying or supplying either one first.

[0022] (3) In the production method according to (1) or (2), in the step (a), the use amount (in moles) of the second organosilane compound for a linker is determined in the step (a). It may be larger than the amount (number of moles) used of one organosilane compound for a linker.

[0023] (4) In the above-described method for producing a probe-immobilizing carrier of the present invention or the production method according to any one of (1) to (3), the step (a) mainly comprises an organic solvent. In the medium (preferably only an organic solvent), the step may be a step of bringing the organosilane compound for a linker into contact with the solid phase substrate.

(5) In the production method according to (4), the organic solvent is one or more selected from toluene, methanol, ethanol, acetonitrile, THF, and chloroform. A little.

[0025] (6) In the above-described method for producing a probe-immobilizing carrier according to the present invention or the production method according to any one of (1) to (5) above, the step (b) includes an organic solvent. The organic silaniy in the main medium (preferably only an organic solvent, preferably one, or two or more selected from toluene, methanol, ethanol, acetate nitrile, THF, and chloroform form force). It is good also as a process of making a compound and the above-mentioned solid phase substrate contact.

[0026] (7) In the above-described method for manufacturing a probe-fixing carrier according to the present invention or the manufacturing method according to any one of (1) to (5) above, the step (b) As an organic silane compound, the interaction or electrostatic interaction between the linker and the linker partial position of the linker is higher than the interaction or electrostatic interaction between the linker partial position and a hydroxyl group. A first organosilane compound for a cabbing having a small functional group and one hydrolyzable group X, and two or more (preferably three) hydrolyzable groups having the functional group. And a second organosilane compound for caving with X. [0027] (8) A method for producing a probe-immobilizing support has a partial linker position capable of binding to a probe or the like and a single hydrolyzable group with respect to a solid phase substrate having a hydroxyl group on the surface. A first organic silane compound for a linker, a second organic silane compound for a linker capable of polycondensation with an organic silane compound having a partial position of the linker and having two or more hydrolyzable groups; And introducing the first and second organosilane compounds into the surface of the solid phase substrate through a bond between an oxygen atom and a key atom. Furthermore, the interaction by hydrogen bonding with the linker partial position of the first organic linker silane compound and Z or the linker partial position of the second linker organic silane compound and Z or electrostatic And a step of introducing an organic silan compound for a cabbing having a functional group whose interaction is smaller than a hydroxyl group into the surface of the solid phase substrate.

[0028] (9) A method for producing a probe-immobilizing carrier includes: a first linker-organosilane compound having a partial linker position capable of binding to a probe or the like and having one hydrolyzable group; An organic silane compound having a linker moiety and having two or more hydrolyzable groups and a second organosilane compound for linker polymerization capable of polycondensation, and supplying the first and second A step of introducing an organic silan compound to the surface of the solid phase substrate through a bond between an oxygen atom and a key atom; and after introducing the organosilane compound, a probe is introduced using the partial position of the linker. It is good also as a thing provided with the process to do. An example of this manufacturing method is shown in FIG. This manufacturing method further includes an interaction by hydrogen bonding with the linker partial position of the first organosilane compound for the first linker and Z or the linker partial position of the second organosilane compound for the second linker. And a step of introducing an organosilane compound for cabbing having a functional group such that Z or electrostatic interaction is smaller than that of a hydroxyl group into the surface of the solid phase substrate.

[0029] (10) In the method for detecting a target compound of the present invention described above, the probe is an oligonucleotide or a polynucleotide having a natural or non-natural base, and the interaction is a base constituting the nucleotide. Hybridization based on hydrogen bonds between them may be used.

[0030] (11) In the above-described carrier for probe fixation according to the present invention, the front of the linker The linker partial position has one or more amino groups and thiol groups selected, or one or more functional groups selected from ester groups, aldehyde groups, epoxy groups and carboxy groups. Also good. Alternatively, the cap may have an alkyl group having steric hindrance as the functional group. Alternatively, the cap may have a monomeric organosilane moiety held on the surface of the solid phase substrate through a bond between an oxygen atom and a key atom. Alternatively, the cap has an organic siloxane moiety or an organic polysiloxane moiety (also referred to as a polymeric organosilane moiety) bonded to the surface of the solid phase substrate through a bond between an oxygen atom and a key atom. Have it. Alternatively, the cap compresses at least one or two or more kinds of an organosilane compound for a cabbing having at least one and three or less hydrolyzable groups X having the functional group on the surface of the solid phase substrate. It may be obtained by polymerization. In addition, the organosilane compound for the cabbage is composed of one that has one hydrolyzable group X, one that has two hydrolyzable groups X, and one that has three hydrolyzable groups X! !, Or 1S Two or more different hydrolyzable groups X may be combined. Further, an organosilane compound having one hydrolyzable group X and an organosilane compound having two or more hydrolyzable groups may be combined. For example, an organic silane compound having one hydrolyzable group X and an organic silane compound having the same three may be used in combination, or an organic silane compound having the same two and an organic silane compound having the same two may be used. The same two organic silane compounds and the same three organic silane compounds may be used, or the same, two and three organic silane compounds may be used in combination. Good. A monomeric cap can be easily obtained by using an organosilane compound having one hydrolyzable group X, and a polymer cap is an organosilane having two or three hydrolyzable groups X. It can be easily obtained by using at least a compound (preferably having three hydrolyzable groups X). Further, on the surface of the solid phase substrate, the organosilane silane compound for cabling is condensed with a hydroxyl group on the surface of the solid phase substrate (or condensed with the hydroxyl group and condensed with the organic silane compound). )

(12) In the above-described probe fixing carrier according to the present invention, the linker is a linker. It may have a hydrocarbon group terminated with one site (for example, an amino group). Here, the hydrocarbon group also has an alkyl group strength of 1 to 10 carbon atoms, preferably a linear alkyl group having 2 to 8 carbon atoms (ethyl group, propyl group, butyl group). Group, pentyl group, hexyl group, hexyl group and octyl group). Further, the linker may have a monomeric organic silane moiety or an organic polysiloxane moiety that is held on the surface of the solid phase substrate through a bond between an oxygen atom and a silicon atom. The monomeric or polymeric linker is at least one or two or more selected from organic silane compounds having one or more and three or less hydrolyzable groups having the linker partial position. It can be obtained by condensation polymerization on the surface of a solid phase substrate. In addition, the organosilane compound for the linker is composed of one that has one hydrolyzable group X, one that has two hydrolyzable groups X, and one that has three hydrolyzable groups X. However, two or more different types of these hydrolyzable groups X may be combined! /. Further, an organosilane compound having one hydrolyzable group X and an organosilane compound having two or more hydrolyzable groups may be combined. For example, an organic silane compound having one hydrolyzable group X and an organic silane compound having the same three may be used in combination, or an organic silane compound having the same number and an organic silane compound having the same number may be used. It is also possible to use the same two organic silane compounds, the same three organic silane compounds, or a combination of the same, two and three organic silane compounds. It may be used. The monomeric linker can be easily obtained by using an organic silane compound having one hydrolyzable group X, and the polymer linker can be obtained by using an organic silane group having two or more hydrolyzable groups X. It can be easily obtained by using a compound. On the surface of the solid phase substrate, the organosilane compound is subjected to polycondensation with a hydroxyl group on the surface of the solid phase substrate (or polycondensation with the hydroxyl group and polycondensation between organosilane compounds).

(13) The probe immobilization carrier is a monomer-like substance held on the solid phase substrate through a bond between a solid phase substrate, a linker part capable of binding to a probe, etc., and an oxygen atom and a key atom. A first linker having an organic silane moiety; a linker moiety capable of binding to a probe; and an organic substance held on the solid phase substrate via a bond between an oxygen atom and a key atom. And a second linker having a siloxane moiety or an organic polysiloxane moiety.

[0033] (14) The probe immobilization carrier includes a first organosilane site having a monomeric organosilane moiety held on the surface of the solid phase substrate via a bond between an oxygen atom and a key atom on the solid phase substrate. It may have a cap and a second cap having a polymer-like organosilane moiety held on the surface of the solid phase substrate through a bond between an oxygen atom and a key atom.

[0034] (15) A DNA array, the probe immobilization carrier produced by any of the production methods described above or any of the probe immobilization carriers described above, and the probe immobilization carrier And a probe held via the linker partial position. In this case, the probe may be an oligonucleotide or a polynucleotide which may have a natural or non-natural base! /.

Brief Description of Drawings

FIG. 1 is a scheme showing an example of a method for producing a probe fixing carrier.

FIG. 2 is an explanatory view showing a carrier for immobilizing a probe, where (a) shows an example when the cap is of a type, and (b) shows an example when the cap is of a plurality of types.

FIG. 3 is an explanatory view showing an example of a monomeric cap.

FIG. 4 is an explanatory view showing an example of a polymer cap.

FIG. 5 is an explanatory view showing an example in which the first and second linkers are mixed in the probe fixing carrier.

FIG. 6 is an explanatory diagram showing an example in which two types of linkers having a monomeric organosilane moiety are mixed in the probe fixing carrier.

FIG. 7 is an explanatory view showing an example of a linker having an organic siloxane moiety (or an organic polysiloxane moiety).

FIG. 8 is an explanatory diagram showing a DNA array, where (a) shows an example of using a linker having an organic siloxane moiety (or an organic polysiloxane moiety), and (b) shows an organosiloxane moiety ( Alternatively, an example in which a linker having an organic polysiloxane moiety) and a linker having a monomeric organic silane moiety are used in combination is shown.

FIG. 9 is an explanatory view showing an example of a first and second organic silane compound for a linker. FIG. 10 is an explanatory view showing an example of a method for producing a probe-immobilizing carrier using the first and second linker-specific organosilane compounds.

FIG. 11 is an explanatory diagram of oxanosin.

FIG. 12 is a graph showing the fluorescence intensity measured in Example 1.

FIG. 13 is a graph showing the fluorescence intensity measured in Example 2.

BEST MODE FOR CARRYING OUT THE INVENTION

[0036] The above-described method for producing the probe-fixing carrier according to the present invention will be described in further detail.

[0037] (Solid phase substrate and linker)

The solid phase substrate is not particularly limited, and various conventionally known carriers used in the hybridization assembly can be used. Examples thereof include polymers such as glass, ceramics, polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, and polymethyl methacrylate, porous glass, porous ceramics, porous silicon, short fibers, and membrane filters. Preferred examples of the solid phase substrate to which the present invention is applied include those in which hydroxyl groups are originally present on the surface, and those in which hydroxyl groups are generated on the surface by performing some kind of treatment on the surface. Specific examples include a solid phase substrate having a silanolic hydroxyl group derived from the material, and a solid phase substrate to which surface hydroxyl groups have been imparted, increased or activated by surface treatment or chemical treatment with plasma or the like. . Particularly preferred is a solid substrate having at least a surface layer of glass, silicone polymer, or silicon.

The form of the solid phase substrate is not particularly limited as long as it has a surface (including an inner surface) to which the probe can be immobilized and the target nucleic acid can be supplied. For example, a flat plate shape such as a substrate, a particle shape such as a bead, a fiber shape, a capillary shape with an inner surface as a fixed surface can be employed.

[0039] The solid phase substrate in the present invention has a linker having a linker site on the surface of the solid phase substrate for fixing the probe. One preferred form of the linker moiety has active hydrogen. For example, the linker partial position contains an amino group or a thiol group. The amino group and the thiol group are the ester group, aldehyde group, and epoxy group provided in the probe. And a covalent bond is formed with the carboxy group to immobilize the probe to the solid phase substrate. The amino group may be an unsubstituted amino group or a substituted amino group. One or two substituents in the substituted amino group are each a hydrocarbon group that may be an independent hydrocarbon group, preferably having 1 to 18 carbon atoms, and preferably having 1 to 6 carbon atoms. A chain alkyl group is more preferred. The amino group is preferably a primary amino group considering the reactivity with the linker compound.

[0040] Other preferred forms of the linker partially include an ester group, an aldehyde group, an epoxy group, and a carboxy group. These functional groups form a covalent bond with an amino group or a thiol group provided in the probe to fix the probe to the solid phase substrate.

[0041] Such a linker can also be used as various mono-, di- and tri-alkoxy silanes having mono-linkers such as amino groups or mono-, di- and trihalogenated silanes (organic silane compounds for linkers). In that case, the silicon atom forms a siloxane bond with the oxygen atom of the silanol group existing on the surface of the glass solid phase substrate. A linker moiety such as an amino group is preferably designed to be provided at the end. It is preferable that an appropriate linking moiety is present between the amino group and the key atom. Such a linking moiety is preferably an alkyl group having 2 to 10 carbon atoms or a form in which some carbon atoms of the alkyl group are substituted with nitrogen atoms or S atoms. The alkyl group can be linear or branched, but is preferably linear. The organosilane compound for a linker is preferably provided with 1 to 3 alkoxy groups and halogen groups as hydrolyzable groups.

[0042] Further, examples of the alkoxy group of the alkoxysilane in the organic silane compound for a linker include a methoxy group, an ethoxy group, an n propoxy group, an isopropoxy group, an n butoxy group, a sec butoxy group, and a tert butoxy group. . The remaining group in alkoxysilane (group bonded to Si in addition to alkoxy group, amino group and alkylamino group) is preferably an alkyl group. As the alkyl group, an alkyl group having about 1 to 10 carbon atoms is preferably used, and a methyl group, an ethyl group, an n propyl group, an isopropyl group, an n butyl group, a sec butyl group, a tert butyl group, an n-hexyl group. Group, cyclohexyl group, n-octyl group, tert-octyl group and n-decyl group. In addition, on the solid phase substrate As the alkoxysilane introduced into the hydroxyl group, various other silane compounds can be used without being limited thereto. Examples of silane compounds that can be used for the carrier are shown below.

[Chemical 3]

one

[0043] In the above description, a halogen group may be used instead of an alkoxy group. An alkoxy group and a halogen group are hydrolyzable groups.

[0044] (Cap)

As long as the hydroxyl group on the surface of the solid phase substrate is protected, any kind of cabbing agent may be used for the solid phase substrate for probe immobilization of the present invention. For example, it is preferable to be capped by an organosilane compound having a hydrocarbon group such as a methyl group, an ethyl group, an n-propyl group, an isobutyl group, or an n-butyl group on a silicon atom. In order to give such a cap to the hydroxyl group on the surface of the solid phase substrate, an organosilane compound having a hydrolyzable group and having at least one hydrocarbon group can be used. Such an organic silane compound preferably has a lower alkoxy group such as a methoxy group or an ethoxy group or a hydrolizable group as a hydrolyzable group on a silicon atom. The number of hydrolyzable groups is not particularly limited. However, when an organic silane compound having one hydrolyzable group is used, the organic silane compound is bonded to the surface of the solid phase substrate in the form of a monomer. Get cap It is done. If an organosilane compound having two hydrolyzable groups is used, a linear or other polymer-like organic metalloxane moiety or organic polymetalloxane moiety linked to the surface of the solid phase substrate is obtained, and three or more When an organic silane compound for a cabbing having a hydrolyzable group is used, a polymer-like organic metalloxane moiety or an organic polymethyloxane moiety such as a network can be obtained. An organosilane compound having one hydrolyzable group (for example, a methyl group or an ethyl group is preferred as a functional group) and two or more (preferably three or more) hydrolyzable groups (preferably propyl By using an organic silane compound having a group or an n-butyl group (more preferably a butyl group or more), the surface hydroxyl group of the solid phase substrate can be efficiently or highly efficiently probed. .

[0045] (probe)

Probes are used for hybridization of nucleic acids, ie oligonucleotides or polynucleotides. In the present invention, the nucleic acid may be any nucleic acid that has at least a base pairing region and hybridizes with other nucleic acids through the pairing region. Therefore, nucleic acids include both natural and synthetic nucleotide oligomers (oligonucleotides) and polymers (polynucleotides), as well as genomic DNA, DNA such as cDNA, PCR products, RNA such as mRNA, and peptide nucleic acids. It is a concept. Here, the term “oligonucleotide” mainly means a polymer having several to preferably several hundred nucleotides, more preferably about one hundred, and even more preferably several tens of nucleotides. Includes those obtained by synthesis. Polynucleotide mainly means a polymer having a longer chain than an oligonucleotide, and includes PCR products and the like.

[0046] The probe is preferably an oligodeoxyribonucleotide having polyoxyribose or a polydeoxyribonucleotide.

[0047] (Linker unit)

The probe used in the present invention may have a linker unit that can be covalently bonded to a portion of the linker provided on the surface of the solid phase substrate. When the amino group or the thiol group is a partial linker, it is preferable that the linker unit contains an ester group, an aldehyde group, an epoxy group, and a carboxyl group, an ester group, an aldehyde group, an epoxy group, and a force. When the lupoxy group is a partial linker position, the linker unit preferably contains an amino group or a thiol group.

[0048] In addition, for a linker partial position having an active hydrogen such as an amino group, for example, a linker compound described below is preferably provided as a linker unit. It is preferable that this linker unit be provided in the probe as a nucleotide derivative like other monomer units constituting the probe.

[0049] Hereinafter, the linker compound will be described. The linker compound exemplified here is a compound represented by the formula (9). Further, a preferred form contained in the probe as a linker unit is shown in Formula (10). A may be a hydrogen atom, other substituents, saccharides or derivatives thereof. When A is hydrogen, the compound represented by formula (9) is an oxanine base. Oxanine base is guanosine induced by NO or NO.

2

It is one of the damaged bases formed by acid deamination. Hereinafter, in the present specification, a nucleoside form in which oxanine is bound to ribose or deoxyribose (both are also furanose type) is referred to as oxanosin, and in particular, deoxyribonose is referred to as deoxyxanosine. To do.

[Chemical 4]

[Wherein, A represents a hydrogen atom, another substituent, a saccharide or a derivative thereof. ] [Chemical 5]

[Wherein, X represents a hydrogen atom, a hydroxyl group, or a substituent. ]

[0050] Oxanine, for example, reacts deoxyguanosine with NO and Z or NaNO

2

Can be obtained as deoxyxanosine. For example, NaNO in doxyguanone under acidic conditions, for example, using sodium acetate buffer (3.0 M, pH 3.7), 3

2

It can be obtained by reacting at 7 ° C for 4 hours. After such a reaction, the reaction solution is neutralized with NaOH, and after appropriately distilling off under reduced pressure as necessary, it can be purified by reverse phase chromatography or the like and dried to obtain deoxyxanosine as a white powder. it can. The mobile phase can be well separated by using a mixture of neutral buffer such as sodium phosphate buffer (400 M, pH 7.4) and organic solvent such as acetonitrile (acetonitrile 10%, etc.). Oxanine can be fractionated by reverse-phase chromatography, etc., based on acid hydrolysis of deoxyxanosine and the reaction solution. For the acid hydrolysis, for example, a reaction condition of 2 hours at 37 ° C. with an acid such as 1M hydrochloric acid can be employed.

[0051] The A may be a saccharide such as pentose such as ribose or deoxyribose. Derivatives of saccharides are those having functional groups such as alkoxy, alkoxyalkyloxy, and halogen instead of hydrogen or hydroxyl groups bonded to carbon atoms of saccharides. Can be mentioned.

[0052] The linker compound of the formula (9) comprises oxanine in any form as long as the reactivity with the amino group is ensured over the carboxy group activated with carpositimide. It's okay. Preferably, it is in a form bound to pentose such as ribose or deoxyribose. Further, the bonding position between the oxanine base and the pentose is preferably in a form in which the C1′-position carbon atom of the pentose is bonded to the N-position at the 3-position. When the pentose is 1 13 ribofuranosyl ribose or ribofuranosyl deoxyribose, the linker compound will take the form of a nucleoside derivative. As already explained, a linker compound in the form of a nucleoside derivative can easily obtain a deoxyguanine or deoxyguanosine force.

Such a linker compound in the form of a nucleoside derivative may be subjected to various modifications suitable for obtaining an oligonucleotide by chemical synthesis. For example, the C3, position hydroxyl group or C5, position hydroxyl group of ribose or deoxyribose is appropriately modified. It is preferable that a phosphoramidite group is introduced into the hydroxyl group at the 3-position, and a dimethoxytrityl group or the like is introduced into the hydroxyl group at the 5′-position. As such a modifying group, known methods can be used as appropriate in the chemical synthesis method of nucleic acids. Examples of linker compounds suitable for chemical synthesis of nucleic acids by the phosphoramidite method are shown below.

[Chemical 6]

[Wherein R ¹ represents 2 cyanoethyl, 4-trophenyl-ethyl, 2 trimethylcytylethyl, or methyl] [0054] Such a linker compound can be obtained by applying a known method for synthesizing monomers for nucleic acid synthesis. For example, after adding dimethoxytrityl chloride to a suspension of deoxyxanosin (such as DMF as a solvent), add imidazole and diisopropylethylammum mesylate, and stir the reaction mixture to homogeneity. Then, it is poured into a large amount of water, a precipitate is obtained, and dried to obtain a crude product. This crude product is redissolved in an appropriate solvent such as dichloromethane and then recrystallized in hexane to obtain DMT-deoxyxanosine.

[0055] Further, in order to phosphoamiditeize DMT-deoxyxanosine, DMT-deoxyxanosine is dissolved in an appropriate solvent, and then 2-cyanoethyl-N, N, Ν ', Ν, After adding diisopropylphosphoamide and allowing it to react sufficiently, the reaction is terminated by adding methanol, diisopropylethylamine and ethyl acetate, washing with 10% sodium bicarbonate, and then dehydrating and concentrating the organic phase. did. This concentrate was redissolved in an appropriate solvent such as dichloromethane and separated by normal phase chromatography. As the mobile phase, for example, ethyl acetate: dichloromethane: methanol = 65: 35: 0.33) can be employed.

[0056] Further, such a linker compound preferably takes the form of a phosphate ester to the hydroxyl group bonded to the carbon atom at the 5-position of ribose or deoxyribose. The phosphate ester is preferably a triphosphate ester. In this case, the linker compound can be taken in the form of a nucleotide derivative. Such a probe compound can be preferably used for obtaining a nucleic acid such as a long-chain DNA of 100 bases or more by a gene amplification technique such as PCR. An example of a linker compound as a nucleotide derivative is shown in the following formula.

[Chemical 7]

An oligonucleotide containing a linker unit that is a nucleotide derivative is known. The probe manufacturing method can be applied. When using a DNA chain extension method using various DNA polymerases, for example, deoxyxanosin triphosphate (dOTP) is prepared as a linker compound. Oxanine is presumed to form stable base pairings by forming two hydrogen bonds with cytosine thymine (preferably cytosine). In consideration of such base pairing, prepare a desired template and a primer from the 5 'end to the desired position to introduce a linker compound, and perform the first extension step in a reaction system containing only dOTP. carry out. Thereafter, the remaining dOTP is removed, and the second and subsequent extension steps are carried out in the presence of the necessary dNTP (N is one or more of A, T, C and G) as usual. . Then, if necessary, an extension step of only dOTP can be carried out to introduce the present linker compound into two or more desired sites.

[0058] In addition, in the case of the phosphoamidite method, for example, the phosphoamidite derivative of deoxyxanosin described above is prepared, and the desired order of introduction with respect to the oligodeoxynucleotide being synthesized. To add a chain extension reaction. This makes it possible to introduce the present linker compound into one or more desired sites of the probe.

[0059] Although this probe functions in the form of a single-stranded nucleic acid in hybridization, it may be in a double-stranded form at the time of acquisition, synthesis, or fixation. Moreover, when taking the form of a single strand, it may have a loop or a hairpin part.

[0060] The probe may comprise one or more linker units. The linker can be provided at the 5 ′ end, which is a carrier fixing site exclusively selected in the probe, or can be provided at other sites such as the 3 ′ end. For example, it may be provided near the center of the entire length of the probe. In addition, when a linker cue is provided on the 5, end side or on the 3, end side, and such a probe is fixed to a carrier, a part of the probe containing the linker compound portion is located upstream and downstream. It can also be designed to have a hairpin structure or loop structure. In particular, when the linker unit takes the form of a nucleotide derivative, the linker can be introduced at an arbitrary site by chemical synthesis or by enzymatic chain extension as already described.

[0061] In this probe, the hybridizing region with the target nucleic acid is a linker unit. It is preferably not included. Therefore, when a linker unit is introduced near the center or near the end of the entire length of the probe, it is preferable to form a spacer region not involved in hybridization in the vicinity.

[0062] In addition, such a spacer region is the same even when a linker unit is introduced at the 5th end, etc. of the probe. It is preferable that the region is a hybrid region.

[0063] (DNA array)

A DNA array is a probe immobilized on a probe immobilization carrier. Here, the probe is a linker compound (a compound for binding the probe to a partial position of the linker on the solid phase substrate or a site where the compound is introduced) and a carboxy group activated with calpositimide of oxanine and a carrier surface. It is preferable that the amino group is fixed in a state in which it reacts with an amino group to form an amide bond.

[0064] The immobilization form of the probe differs depending on the position of the linker compound introduced into the probe. When the linker compound is provided at the 5 'end, the probe will be immobilized in a single strand, If a linker compound is provided near the center of the entire probe length, the probe will be immobilized in a U-shape. In addition, in the case of having a sequence that can form base pairing in the vicinity of the vicinity of the linker compound introduction site, a hairpin or loop structure will be adopted in the vicinity of the linker compound introduction site.

[0065] (Method for producing DNA array)

In the DNA array, a liquid containing a probe is prepared, and this liquid is supplied to the surface of the above-mentioned probe fixing carrier, and the linker compound oxanin in the probe and the amino acid provided in the probe fixing carrier are prepared. It can be obtained by reacting with a group or the like. As a method for supplying the probe to the probe-immobilized carrier, a conventionally known deviation method can be employed. For example, in order to produce an array in which a large number of probe spots are arranged on the surface of a substrate or the like as a DNA array, probe-containing droplets are supplied in the form of spots by a contact type using a pin or an inkjet method. Further, the probe can be supplied to particles such as beads by dipping the beads in the probe-containing solution or spraying the probe-containing solution. [0066] Immobilization of the probe can be carried out by such an operation as spotting of the probe solution, which is usually performed, and dating. If the probe solution is supplied to the probe-immobilized carrier having an amino group, the linker compound and the amino group can react rapidly. In addition, an organic solvent can be added to the probe solution, and it can be adjusted to neutral or slightly acidic as necessary. The temperature and incubation time may be appropriately set according to the type of probe immobilization carrier or probe. For example, the temperature is set to a predetermined temperature of 30 ° C to 90 ° C, and the reaction time is from several tens of minutes to 1 After supplying the probe solution to the surface of the probe-immobilized carrier, which can be made for several hours or several hours, steps such as reno, idling, blocking, denaturing, washing and drying can be carried out.

[0067] (Hybridization method)

The nucleic acid hybridization method of the present invention prepares the above-described DNA array, supplies the nucleic acid to this DNA array, and hybridizes the probe and the target nucleic acid. In this hybridization method, except that the probe immobilization carrier of the present invention is used, various target nucleic acids prepared by a conventionally known method may be supplied and hybridized according to a conventional method.

[0068] This specification includes the entire contents of Japanese Patent Application No. 2005-065948, filed in Japan on March 9, 2005, by reference. This application is based on 60Z749, 590, which was provisionally filed in the United States on December 13, 2005, and claims the priority thereof, the entire contents of which are incorporated herein by reference.

Example 1

[0069] A probe-fixing carrier was prepared, and a confirmation test of the cabbing effect was performed. The procedure is described below. Here, a non-coated slide glass for DNA microarray (manufactured by Matsunami Glass Industrial Co., Ltd.) was used as a solid phase substrate (hereinafter referred to as substrate). APS is an abbreviation for 3-aminobutylpyrutriethoxysilane, and is an example of an organosilane compound for a linker. BMS is an abbreviation for ptyltrimethoxysilane, and is an example of an organosilane compound for caving.

[0070] (1) Modification of substrate surface

First, the substrate surface was modified. That is, 20 substrates are 10 in 800 mL acetone. After ultrasonic treatment for 5 minutes, it was vacuum dried in a desiccator for 10 minutes at room temperature. Subsequently, 20 of these substrates were added to 1200 mL of parahana solution (840 mL of 90% H 2 SO 4 and 360 mL of

twenty four

Soak in 30% H 2 O and a solution that can also be sonicated for 30 minutes, then 80 mL of distilled water

twenty two

After soaking in water and treating with ultrasonic waves for 10 minutes, it was vacuum dried in a desiccator at room temperature for 1 hour. The substrates were again immersed in 800 mL of distilled water and treated with ultrasound for 10 minutes, then vacuum-dried at room temperature for 12 hours in a desiccator, immersed in 800 mL of methanol and treated with ultrasound for 10 minutes, and then treated with 800 mL of Immerse in a 1: 1 (volume ratio) mixture of methanol and toluene (400 mL of methanol and 400 mL of toluene) and treat with ultrasound for 10 minutes, then soak in 80 OmL of toluene for 10 minutes with ultrasound. Processed. 20 substrates that have been treated in this manner are immersed in 1290 mL of 2% APS solution (25.8 mL of APS and 126.4.2 mL of toluene) and rocked at 70 rpm in an incubator for 5 hours. Reacted. Then, the substrates are immersed in 800 mL of toluene and treated with ultrasound for 10 minutes, immersed in 800 mL of a 1: 1 mixture of methanol and toluene (volume ratio), treated with ultrasound for 10 minutes, and further treated with 800 mL. The sample was immersed in methanol and treated with ultrasonic waves for 10 minutes, and then dried in a constant temperature dryer at 110 ° C. for 1 hour. The substrates were shielded from light and vacuum-dried in a desiccator at room temperature for 12 hours. Ten substrates out of these treatments were stored as APS-modified substrates (hereinafter referred to as APS substrates).

[0071] On the other hand, the remaining 10 substrates after such treatment were immersed in 1200 mL of 2% BMS (caping agent) solution (24 mL of BMS and 1176 mL of toluene), and in an incubator at 25 ° C and 70 rpm. The reaction was performed for 5 hours while shaking. The substrates are then immersed in 800 mL of toluene and treated with ultrasound for 10 minutes, then immersed in 800 mL of a 1: 1 mixture of methanol and toluene (volume ratio), treated with ultrasound for 10 minutes, and then further treated with 800 mL of toluene. After immersion in methanol and ultrasonic treatment for 10 minutes, drying at 110 ° C for 1 hour in a constant temperature drier, further drying these substrates in a desiccator and vacuum drying at room temperature, and modifying them with APS In addition to the BMS-capped substrate (hereinafter referred to as APS + BMS substrate)

[0072] (2) Probe spotting and immobilization

Subsequently, the oligo DNA probe was spotted and fixed. Here, Ori As a DNA probe, one having a sequence of Chemical Formula 8 was used. In the sequence, “0” at the 5th end represents oxanosin, “TTT” following it represents a spacer, and the remaining sequence following the spacer, ie “CAG '· -ACGJ (dotted line underline) Represents the complementary sequence of the hybridization sample.

[Chemical 8]

5 '-0 TTT CAGCA CCAAT GTCCT TGGCC ATCTT CAACT TGTTG TCGAA AATGT CAACG-3'

[0073] Spotting and immobilization were carried out by the following procedure for two APS substrates and two APS + BMS substrates. First, the oligo DNA probe was dissolved in PBS, and the oligo DNA was spotted at a concentration of 50 pmol Z μL using a microceramic pump type DNA chip production apparatus GENESHOT (manufactured by NGK). Next, 42. C, incubated overnight at 45% relative humidity, placed the substrate in a glass rack, and immersed in a succinic anhydride solution for 20 minutes. Here, a succinic anhydride solution was prepared by dissolving 2.5 g of succinic anhydride in 160 mL of 1-methyl-2-pyrrolidinone and adding 17.5 mL of 0.2 M aqueous sodium borate (pH 8.0). Then, the slide rack was soaked in sterilized water and swirled gently 10 times, and then washed twice with 2 X SSC, 0.2% SDS aqueous solution at room temperature for 15 minutes, and boiled. It was immersed in 2 X SSC, 0.2% SDS aqueous solution for 5 minutes. Thereafter, the whole slide rack was immersed in sterile water and the operation of gently rocking and washing 10 times was repeated 3 times, followed by centrifugal drying at lOOOrpm for 3 minutes. As a result, two APS substrates and two APS + BMS substrates each having an oligo DNA probe immobilized thereon were obtained.

[0074] (3) Hybridization

Of the two APS + BMS substrates to which the oligo DNA probe was immobilized, one was immediately subjected to the hybridization (0 days after immobilization), the other one was 42 ° C, relative humidity 50 Hybridization was performed after exposure for 7 days under 7% conditions (7 days after fixation). Also, hybridization was performed in the same manner for the two APS substrates. For the hybridization, the oligo DNA shown in Chemical formula 9 was used as a hybridization sample, which was dissolved in sterilized water and used. The oligo DNA of 匕 9 has a complementary sequence (dotted line underlined part) to the above oligo DNA probe and is labeled with Cy3 at the end. [Chemical 9]

Five'

[0075] Hybridization was performed according to the following procedure. First, it was prepared Roh, Eve Lida I See Chillon Sanpunore [This ^{_{20 X SSC, 10 0/0}} SDS mosquitoes卩免, a final concentration of ^{5 X SSC, 0. 5 0/} 0 SDS [Konaru so on. The final concentration of the hybridization sample at this time is InM. Next, 25 μl of the sample solution was dropped on the substrate, and a cover glass was placed on the substrate and incubated at 42 ° C at 100% relative humidity. Next, shake with 2 X SSC, 0.1% SDS solution at room temperature for 5 minutes, shake with 1 X SSC solution at room temperature for 5 minutes, and shake with 0.1 X SSC solution at room temperature. After 5 minutes of shaking at 100 rpm, it was centrifuged at lOOOrpm for 3 minutes. In this way, after performing hybridization, the numerical value of the fluorescence signal was measured for each substrate. The fluorescence signal of Cy3 was measured using a DNA microarray scanner (G2505A, manufactured by Agilent Technologies). The fluorescence signal of each spot was numerically calculated with GenePixxPro (manufactured by Axon Instrument). The result is shown in FIG.

[0076] As is clear from FIG. 12, when the hydroxyl group remaining on the substrate surface after APS treatment was capped with BMS (APS + BMS), the fluorescence signal intensity after 7 days from fixation was In the case of strong force (APS) compared to 0 days after fixation, the fluorescence signal intensity after 7 days after fixation was about 0 days after fixation. Depressed to around 60%.

Example 2

[0077] A probe-fixing carrier was prepared, and a confirmation test of the cabbing effect was performed from a viewpoint different from that in Example 1. The procedure will be described below. Also in this example, the same non-coated slide glass for DNA microarray (manufactured by Matsunami Glass Industrial Co., Ltd.) was used as the substrate.

[0078] (1) Modification of substrate surface

The same operation as in Example 1 was performed.

[0079] (2) Probe spotting and immobilization

Subsequently, the probe was spotted and immobilized. Here, Cy3 Mono -reactive Dye Pack (PA23001, manufactured by GE Healthcare Nanoscience) is used as a probe. used. Spotting and fixing were performed by the following procedure for each APS substrate and APS + BMS substrate. First, Cy3 was spotted at a concentration of 50 pmol Z μL using a micro ceramic pump type DNA chip production apparatus GENESHOT (manufactured by NGK). Subsequently, incubation was performed at 42 ° C. and a relative humidity of 45%. The substrate was placed in a slide glass rack and immersed in a succinic anhydride solution (described above) for 20 minutes. Then, the slide rack was soaked in sterilized water and swirled gently 10 times. After 2 times of washing, it was shaken and washed with 2 X SSC, 0.2% SDS aqueous solution at room temperature for 15 minutes and boiled. It was immersed in 2 X SSC, 0.2% SDS aqueous solution for 5 minutes. Then, the slide rack was soaked in sterilized water 10 times, and the operation of gently rocking and washing was repeated 3 times, followed by centrifugal drying at lOOOrpm for 3 minutes. As a result, one APS substrate and one APS + BMS substrate on which Cy3 was fixed were obtained.

[0080] (3) Pseudohybridization

Pseudohybridization was performed using the APS + BMS substrate and APS substrate to which Cy3 was immobilized (immediately after fixation). The pseudo-hybridization was performed according to the following Tagawa page. First, mix 20 X SSC and 10% SDS, and adjust to a final concentration of 5 X SSC and 0.5% SDS, and add this to the DNA! Pseudohybridization sample. The liquid was used. Next, 25 1 of the sample solution was dropped on the substrate, and the top force was also applied to a cover glass, and incubated at 42 ° C at 100% relative humidity. Next, shake with 2X SSC, 0.1% SDS solution at room temperature for 5 minutes, shake with 1X SSC solution at room temperature for 5 minutes, and further shake with 0.1X SSC solution at room temperature. The mixture was shaken for 5 minutes and then centrifuged and dried at lOOOrpm for 3 minutes. After performing pseudo-hybridization in this way, the fluorescence signal was digitized for each substrate. The fluorescence signal of Cy3 was measured using a DNA microarray scanner (G2505A, manufactured by Agilent Technologies). Moreover, the fluorescence signal of each spot was quantified with Gene PixxPro (manufactured by Axon Instrument). The results are shown in FIG.

[0081] As is apparent from FIG. 13, the fluorescence intensity is significantly increased when the hydroxyl group remaining on the substrate surface after APS treatment is capped with BMS (APS + BMS) and when it is not capped (APS). did. In Example 1, the fluorescence intensity of both on the 0th day after immobilization was such that the APS + BMS substrate was slightly increased compared to the APS substrate. The sample is different between Example 1 and Example 2. It seems to be caused by that.

[0082] Further, in Example 2, water remaining on the surface of the substrate after being treated with APS using ETMS, MTPS, ODTES, EMS, MMS (see the above-mentioned Chemical Formula 3) instead of BMS. The acid group was protected, and pseudo-hybridization was performed in the same manner, and the fluorescence signal was measured. The results are shown in FIG. As is clear from FIG. 13, the fluorescence intensity increased for all the substrates protected with the remaining hydroxyl groups compared to the APS substrate.

Industrial applicability

[0083] The present invention can be used in bio-related technologies related to the production and use of DNA arrays.

Claims

The scope of the claims

[1] (a) A solid phase substrate having a hydroxyl group on the surface is reacted with one or more kinds of organosilane compounds for a linker having a partial linker position capable of binding to a probe, and the oxygen atom of the hydroxyl group is A step of holding the organic silane compound for the linker as a linker on the surface of the solid phase substrate by bonding a key atom of the organosilane compound for the linker, and (b) the (a) A step of protecting a hydroxyl group remaining on the surface of the solid phase substrate after the step with a caving agent;

A method for producing a probe immobilization carrier comprising:

[2] In the step (b), one or two or more kinds of organosilane silane compounds for cabbing that do not have the linker partial position as the cabling agent are used, and the solid after the step (a) is used. Bonding the oxygen atom of the hydroxyl group remaining on the surface of the phase substrate to the silicon atom of the organosilane compound for caving.

The method for producing a probe-immobilizing carrier according to claim 1.

[3] (a) One kind or two or more kinds of organic silane compounds for a linker having a partial linker position capable of binding to a probe with respect to a solid phase substrate having a hydroxyl group on the surface are converted into oxygen atoms of the hydroxyl group. And the step of introducing via a bond with a silicon atom of the organosilane compound for the linker,

A method for producing a probe immobilization carrier comprising:

[4] The production of the probe-immobilizing support according to claim 2 or 3, wherein the organosilane compound for cabbing is a compound having an alkoxy group or a halogen group in addition to an alkyl group on a key atom. Method.

[5] The probe organosilane compound according to any one of [2] to [4], wherein the cabbing organosilane compound is one or more compounds represented by the following formula (1): Production of carrier Method.

SiX R

m n

The probe organic compound according to any one of claims 2 to 4, wherein the organosilane compound for caving is one or more compounds selected from the group consisting of the following formulas (2) to (8): A method for producing a support for stabilization.

oso. — IJ i \

BMS

關 S

ETMS

[7] The probe according to any one of [1] to [6], wherein the organosilane compound for a linker has an alkoxy group and a group having the linker moiety at the end on a silicon atom. A method for producing an immobilization carrier.

[8] The organosilane compound for a linker is one or two or more groups selected from the group consisting of an amino group, a thiol group, an ester group, an aldehyde group, an epoxy group, and a carboxy group as the linker partial position. The method for producing a probe-immobilizing carrier according to claim 1, comprising:

[9] The solid phase substrate according to claim 1 to 10, which is made of glass, plastic or silicon. A method for producing a probe fixing carrier according to any one of the above.

[10] By introducing the probe into the probe solid substrate carrier produced by the method for producing a probe-fixing carrier carrier according to any one of claims 1 to 9, using the partial position of the linker. A method for producing a DNA array, wherein a DNA array is obtained.

[11] The target compound is detected by the probe by supplying a test sample containing a target compound that interacts with the probe to the DNA array according to claim 10, or by the probe Capturing the target compound and detecting a signal that changes or is generated by the interaction between the probe and the target compound.

A method for detecting a target compound.

[12] A probe immobilization carrier produced by the method for producing a probe immobilization carrier according to any one of claims 1 to 9.

[13] a solid phase substrate;

One or two or more types of linkers that are held on the surface of the solid-phase substrate by binding a carbon atom to an oxygen atom on the surface of the solid-phase substrate that has a partial linker position that can bind to the probe. When,

A cap protecting oxygen atoms on the surface of the solid phase substrate to which the linker is not bonded;

A probe immobilization carrier comprising:

[14] a solid phase substrate;

One or two or more types of linkers which are held on the surface of the solid phase substrate and have a partial linker position capable of binding to a probe;

One or two or more kinds of caps having a hydrocarbon functional group and held on the surface of the solid phase substrate through an oxygen atom and a key atom bond;

A probe immobilization carrier comprising:

[15] The cap has a hydrogen bond interaction or electrostatic interaction with the linker partial position of the linker, or a hydrogen bond interaction or electrostatic interaction between the linker partial position and a hydroxyl group. 15. The probe immobilization carrier according to claim 14, which has a smaller functional group.