JP2003322653A - Support and carrier for fixing probe - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus capable of highly reliably detecting substances related to living bodies. <P>SOLUTION: The probe fixing support is provided with a support; long and narrow carriers of inorganic matter extended to one surface of the support via at least their one ends; and probes fixed to the long and narrow carriers to be specifically combined with target substances. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a biologically relevant substance, for example, a nucleic acid, which utilizes a biospecific bond.
The present invention relates to a device for detecting proteins, antigens, antibodies and the like.

[0002]

2. Description of the Related Art At present, a technique for detecting a target bio-related substance by utilizing a biological specific binding is widely known. For example, probe D for detecting a specific DNA
Examples thereof include a DNA chip in which NA is immobilized on a substrate, a method of detecting an antigen or an antibody using an antigen-antibody reaction, and the like.

In particular, a DNA chip has been attracting attention as a sensor for detecting a nucleic acid capable of easily detecting a specific sequence in basic research fields such as post-genome research and clinical fields such as genetic testing.

A conventional DNA chip is a glass of several cm square.
Or 10 on the surface of the silicon substrate ¹-10⁵Kind of nucleus
This is a device in which an acid is immobilized as a probe. like this
Nucleic acid fragments contained in the sample when using a simple DNA chip
Is generally analyzed as follows. First,
Tried with fluorescent dyes and radioisotopes (hereinafter referred to as RI)
Label the nucleic acid fragments in the sample and then react them with the probe
Let Alternatively, the nucleic acid fragment in the sample is not labeled and used separately.
The nucleic acid fragment in the presence of the intended labeled oligonucleotide
React with the probe, and sandwich hybridization
Option. Contains a sequence complementary to the base sequence of the probe.
If the nucleic acid fragment is present in the sample,
Signals derived from labels (for example, fluorescence intensity and RI intensity)
Is obtained at a specific site. Furthermore, it is fixed to the substrate
Based on the sequence and position of the probe, the base sequence present in the sample
Can be easily checked.

However, the conventional DNA chip has the following problems. For example, two types of probes having a complementarity to a part of the sequence (for example, the case where the base sequence “AAA” is included in one side and the base sequence “TTT” is included in the other side) are arranged at positions close to each other. Binding occurs between these probes. If such unexpected binding occurs between the probes, the desired hybridization will not be achieved. As a result, the reliability of detection and quantitative analysis decreases. Further, even when a polymerase chain reaction (hereinafter, referred to as PCR) is performed on the DNA chip, the reaction efficiency decreases when such an unexpected bond occurs. The unexpected binding between such probes is
Even if the density of fixed probes is not always overcrowded,
That is, even densities, which are usually considered to be adequate, occur.

Further, if the accuracy of detection is to be increased, the uniformity of the density of the fixed probes becomes a problem. For example, when the probe is fixed by spotting, the density of the probe to be fixed is roughly adjusted by the concentration of the probe solution used for spotting. At this time, even if the concentration of the probe solution is adjusted, it is difficult to control even the degree of dispersion of the probe contained in the dropped probe solution. Therefore, it is difficult to make the probe density constant over the entire fixed surface. The variation in the density of the probe fixed by spotting is one of the factors that reduce the reliability of detection and quantitative analysis.

Further, when considering the improvement of detection sensitivity,
Regardless of the type of fluorescence detection or amperometric analysis used conventionally, D hybridized on the DNA chip
We have to increase the amount of NA. On the other hand, conventional DN
The A chip is a device in which a probe is fixed on the surface of a glass or silicon substrate having a size of several cm. Therefore, there is a limit to the number of probes fixed there. Further, in consideration of the problem of unexpected binding between probes and the appropriate probe density as described above, the conventional technique inevitably increases the size of the DNA chip.

[0008]

In view of the above situation, it is an object of the present invention to provide an apparatus capable of highly reliably detecting a biological substance utilizing a biological specific binding. is there.

[0009]

The above object can be achieved by the present invention as described below. That is, (1) a support, an elongated carrier extending through at least one end thereof on one surface of the support, and a probe fixed to the elongated carrier and specifically bound to a target substance. And (2) a probe-immobilized carrier comprising an inorganic elongated carrier and a plurality of probes immobilized on the elongated carrier and specifically binding to a target substance.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION According to one aspect of the present invention, there is provided an apparatus capable of detecting a target substance of interest by utilizing a biological specific binding. As used herein, the term "biological substance" means ribonucleic acid (RNA), deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), methylphosphonate nucleic acid, oligonucleotides such as S-oligo, cDNA, cRNA, etc. Nucleic acids and nucleic acid analogs such as polynucleotides, peptides, polypeptides and proteins,
And substances related to living bodies such as sugar chains, enzymes, antigens and antibodies, and analogs thereof. The term "biological specific binding" used herein refers to a biological specificity such as hybridization due to complementarity between nucleic acids, binding due to antigen-antibody reaction, and binding between receptor and ligand. It refers to the bond and the bond imitated thereto.

As used herein, the term "target substance" refers to the substance of interest to be detected. Preferably polynucleotides, oligonucleotides, DNA and RN
It is a biologically relevant substance such as a nucleic acid such as A, a peptide, a polypeptide and a protein, a sugar chain (that is, a polysaccharide), an enzyme, an antigen and an antibody, and an analog thereof. These may be naturally occurring substances or artificially synthesized substances.

As used herein, the term "probe" refers to a substance that specifically binds to a target substance. For example,
When detecting a nucleic acid, the probe may be any nucleic acid having a sequence complementary to the base sequence of the target nucleic acid. The term "complementary" as used herein means that it is complementary in the range of 50% to 100%, preferably 100%. Further, for example, when detecting a protein, the probe may be a polyclonal antibody or a monoclonal antibody that specifically recognizes the protein. However, it is not limited to these.

I. Configuration of probe holding substrate Examples of the probe holding substrate of the present invention are shown below.

1. First Mode (1) Configuration As a first mode, FIG. 1 schematically shows an example of a probe fixed support using a plate-shaped substrate as a support. Figure 1A
FIG. 1B is a side view of the probe fixing support, and FIG. 1B is a plan view thereof. As shown in FIG. 1A, the probe fixing support 1 includes a substrate 2, an elongated carrier 3 extending through the one end of the substrate 2, and a probe 4 fixed on the surface of the elongated carrier 3. It is equipped with.

Here, "extends through one end" means that one end of the elongated carrier in the longitudinal direction is in contact with the surface of the support and the other end is generally opposite to the surface of the support. It extends in the direction of.

The elongated carrier and the support may be formed of the same member as one body, or may be formed by joining two or more members together.

The probe 4 may be fixed to the elongated carrier 3 on the solid surface. The solid phase surface may be at least a part of the surface provided on the elongated carrier, and the nucleic acid probe is immobilized on the solid phase surface. The surface of the elongated carrier according to the embodiment of the present invention may include a solid phase region where the probe is immobilized and a non-solid phase region where the probe is not immobilized.

Reference is now made to FIG. 1B. There is a certain distance between the adjacent elongated carriers 3a and 3b arranged on the substrate 2. This distance is fixed by the probe 4
Longer than the total length of.

With the structure of the first aspect, it is possible to prevent unexpected binding or steric hindrance between the probes, even though the probes are maintained at a high fixed density. In the case of the conventional device, when the fixation density of the probe is increased, the distance between the adjacent probes fixed on the solid phase surface is narrowed, so that the adjacent probes interfere with each other and the target to be bound is The binding of the substance to the probe is inhibited. Here, inhibition of such binding is called "steric hindrance".

The first embodiment shown here is an example of the probe fixing support according to the present invention. Therefore, the number of elongated carriers 3 arranged on the substrate 2, the number of probes fixed thereto, and the distance between the probes may be arbitrarily changed.

Further, a distance is set so that the probes fixed to the adjacent elongated carriers 3a and 3b do not come into contact with each other.
It may be between adjacent elongated carriers. Alternatively, the lengths of the probes fixed to the adjacent elongated carriers 3a and 3b may be equal to the distance between the elongated carriers.

According to the first aspect, the elongated carriers 3a and 3b.
Each of the probes fixed to is maintained in an independent state for each elongated carrier.

For example, when nucleic acid is used, the length of the probe may be about 3 bases to about 1000 bases, preferably about 10 bases to about 200 bases.

The type of probe to be fixed may be changed for each elongated carrier. Also, some elongated carriers may be set as one set, and the type of the fixed probe may be changed for each set. For example, when a nucleic acid is used as the probe, its base sequence may be changed for each long and thin carrier, or the base sequence may be changed for each set of long and thin carriers.

(2) Shape and material of the long and thin carrier The term "long and thin" used in the present specification means that the aspect ratio is relatively large. The elongated carrier according to the present invention has, for example, an aspect ratio of about 1 to about 5000, preferably about 1 to about 1000.

The elongated carrier according to the present invention is an inorganic substance,
The substance may be a single crystal, a polycrystal, or an amorphous substance of an organic substance or a metal, or their materials and / or properties may be mixed.

The size of the long and narrow carrier used in the present invention is φ = several nm to several tens of μm, and length = several tens nm to
It is several hundred μm. For example, if the long and narrow carrier is a carbon nanotube, φ = several nm. In the case of Si whiskers obtained by etching Si, φ = several μm and the length is several hundred μm.

The elongated carrier according to the present invention, depending on the material,
It may be formed by a method known per se. For example, it may be formed by a crystal growth method, polymer synthesis, etching, or deposition using pores as a template. Further, when the elongated carrier is made of glass fiber, the elongated carrier may also be drawn by drawing. Can be formed.

For example, when the elongated carrier is a crystal, it is formed from a liquid phase, a gas phase or a solid phase, or a coexisting phase thereof, under a suitable growth environment, using a growth method known per se. It is possible. Such growth methods include, but are not limited to, zone melting method, melt growth method such as Czochralski method and pedestal method, top seed method, liquid phase epitaxy method and high pressure flux method. Physical methods such as solution growth method, vapor deposition method and sputtering method,
Also, a chemical vapor deposition method (hereinafter, referred to as CVD), an organic chemical vapor deposition method (hereinafter, referred to as MOCVD), an atomic layer epitaxy method (that is, ALE method), a chemical transport method, and a VLS growth (Vap).
or-Liquid-Solid growth) and the like. When a crystal is grown by epitaxial growth or the like to form a long carrier, the substrate material used for crystal precipitation may be used as it is as the support of the present invention.

Further, although FIG. 1 shows an example in which a columnar elongated carrier is used, the present invention is not limited to this. The elongated carrier according to the present invention may have a linear (eg, straight, curved or spiral) outer shape, a needle shape, a columnar shape, a dendritic shape, or a fibrous shape, and may have a hollow inside. It may be dense. That is, the appearance may be a long and thin shape.

Here, the "linear" elongated carrier refers to an elongated carrier made of a linear substance. Here, the “linear substance” refers to a substance having a large aspect ratio and a small area of a cross section perpendicular to the longitudinal direction thereof, and preferably the cross section is as close as possible to a point. In other words, it is a substance that looks like a line as a whole because the area of the cross section perpendicular to the longitudinal direction is small. Further, the linear substance may be linear, curved, or spiral, in particular, and
It may be a substance having a shape obtained by combining those shapes. As used herein, a “straight-line” substance refers to a substance that has the appearance of a straight line that can connect two points in the shortest distance. As used herein, a "curved" material refers to a material that is linear in shape and has the appearance of a continuous line where every portion is smoothly curved. As used herein, a “spiral” substance refers to a substance that is linear in shape and that has an appearance such that the whole or a part thereof draws a spiral by itself.

Here, the "needle-like" substance is a substance having an area of a cross section perpendicular to the longitudinal direction larger than that of a linear substance, and the one end in the longitudinal direction gradually becomes thinner toward the other end. Have. Refers to a substance that looks like a "needle." Here, the “columnar” substance refers to a substance whose cross-sectional area perpendicular to the longitudinal direction is larger than that of a needle-like substance, and includes a column and a prism. Unlike the needle-shaped material, the cross section of the columnar material perpendicular to the longitudinal direction is almost the same throughout the whole. Here, the “dendritic” substance is a substance having an appearance such that at least one branch portion projects from the side surface of the main trunk portion made of a linear, needle-shaped or columnar substance. The substance constituting the main trunk portion and / or the branch portion may be a substance selected from linear, needle-like and columnar substances, and a combination thereof.

The "fibrous" elongated carrier is composed of a fibrous substance, and the fibrous substance means a substance in which an aggregate of linear substances extends in a disordered direction.

Further, such an elongated carrier having an elongated shape may be curved and / or bent to form a cotton-like lump. In the case of a cotton-like lump, one end of the elongated carrier may be fixed to the surface of the support, and the remaining part may be entirely or partly cotton-like. Alternatively, when the appearance of the cotton-like lump formed by one elongated carrier is long and thin, one end on the longitudinal direction side of the cotton-like lump may be fixed to the support.

For example, examples of preferred elongated carriers used in the present invention are preferably inorganic substances, preferably crystals, and more preferably whiskers.

The "whisker" used in the present specification basically refers to a single crystal of an inorganic substance, an organic substance and a metal, and the size thereof is about 0.1 μm to several tens μm in diameter, and the length is several μm. The thickness may be 10 μm to several hundreds μm or more, and the aspect ratio is generally about 10 or more.

Examples of whiskers that can be used in the present invention are carbon nanotubes, carbon fibers, and boron whiskers (S. Komatsu, and Y. Moriyoshi, J. Crystal Growt).
h, 108, pp.63, 1991), Si ₃ N ₄ whiskers, Si ₃ N
₄ spiral crystals, diamond columnar crystals (Y.sato, M.Kamo, S
urface and Coating Tech.39 / 40, pp.183, 1989), carbon fiber having a spiral structure (Shuji Motoshima, Masayuki Kawaguchi, Hiroshi Iwanaga, Ceramic Data Book 1991, Industrial Products Technology Association, pp.
111 (1991)), ZnO columnar crystals, magnesium sulfate hydroxide whiskers (Yasushi Yoshida, Shuji Yamaguchi, Gypsum & Lime, No.
216, pp. 281, 1988), gypsum whiskers obtained by dehydration of dihydrate gypsum (Toshi Yasue, Toshiya Takase, Hideyuki Umezawa, Yasuo Arai,
Gypsum & Lime, No.216, pp.295, 1988), Au, Cu, A
linear crystals of metals such as g, Fe, Ni, Co and Pt, N
i, Cu, Fe, Pt, Ti, Au, Al, Ba, G
Metal crystals such as e, V, Ag and Pd, and needle crystals of polyoxymethylene, needle crystals of epoxy resin, needle crystals of unsaturated polyester resin, thiazyl whiskers, diacetylene polymer whiskers and m-chloro. Contains organic crystals such as nitrobenzene whiskers.

Further examples of whiskers are described in "Whisker Technology" by Levitt, and any whiskers described therein are preferably used in the present invention (AP Levitt, Whisk.
er Technology, John Wiley & Sons, 1970).

As described above, the "fibrous substance" is a substance in which an aggregate of linear substances grows in a disordered direction. Such materials include Si ₃ N ₄ fibrous crystals, whiskers made of magnesium sulfate hydroxide (Yoshida, Y., Yamaguchi, S., Gyps).
um & Lime, No.216, pp.281, 1988), and gypsum whiskers by dehydration of gypsum dihydrate (Kazuto Yasue, Toshiya Takase, Hideyuki Umezawa,
Arai Yasuo, Gypsum & Lime, No.216, pp.295, 1988) and the like are mentioned as examples.

Examples of the spiral material include spiral Si ₃ N ₄ fibers and spiral carbon fibers (Motoshima Suji, Kawaguchi Masayuki, Iwanaga Hiroshi, Ceramic Data Book 1991, Industrial Products Technology Association, pp.111). , 1991) and the like.

For example, metal linear crystals are subjected to a reduction method, an agglomeration method from a gas phase, a production from a solid phase, an electrolytic deposition method, a thermal decomposition method, and VLS (Vapor-Liquid-Solid).
It can be formed using a synthetic method such as a method.

The linear metal crystal that can be synthesized by such a reduction method can be formed from Au, Cu, Ag, Fe, Ni, Co, Pt, or the like. Further, metal linear crystals that can be synthesized by a coagulation method from the gas phase are Ni, Cu, F
e, Pt, Ti, Au, Al, Ba, Ge, V and Ag
Etc. P is used for metal linear crystals that can be synthesized by thermal decomposition.
d, Pt and Au.

The whiskers usable in the present invention may be in a state in which impurities are mixed to some extent, or may be partially polycrystalline. The carbon nanotubes may also be graphite that forms a layer on its concentric circles. In this case, the material is polycrystalline as a whole, but such a material is also preferably used in the present invention. By the way, such carbon nanotube graphite also
It is generally recognized by those skilled in the art as whiskers.

The elongated carrier is an electrode or an optical fiber.
It may be made of a material having the function of. In that case, for example, when a nucleic acid probe is selected as the probe, the nucleic acid probe immobilized on the surface specifically binds to the redox current or optical signal of the nucleic acid or to the single-stranded nucleic acid. It can be quantified by measuring a redox current or an optical signal of an electrochemically or optically active substance. That is, when the elongated carrier is an electrode, for example, a redox current derived from a nucleic acid or an intercalating agent is measured and fixed using a measurement system including a potentiostat, a function generator, a recorder, and a computer. It is sufficient to quantify the nucleic acid. When the carrier is an optical fiber, the absorbance is an optical signal derived from the nucleic acid or the intercalating agent bound to the nucleic acid, fluorescence intensity, luminescence, quenching, circular dichroism,
The fluorescence polarization or other optical information may be measured by a measuring device corresponding to each signal. Thereby, the nucleic acid can be quantitatively measured.

(3) Support The "support" used in the present invention is a member for supporting the elongated carrier. The support used in accordance with this embodiment is, for example, an amorphous substrate such as glass and resin, a single crystal substrate such as silicon, SiC and α-Al ₂ O ₃ , a polycrystalline Si substrate, a polycrystalline SiC substrate, or the like. It may be a crystal substrate, a porous substrate, or a substrate known per se used for general DNA chips and the like.

In the probe-immobilized support according to the present invention, the elongated carrier may be formed from the support, and after the support and the elongated carrier are separately manufactured, the support and the elongated carrier are bonded to each other. Alternatively, the elongated carrier may be formed as an integral body simultaneously with the formation of the support.

When the support and the elongated carrier are manufactured separately, the probe may be fixed to the elongated carrier before or after the elongated carrier is bonded to the support. When the long carrier and the support are manufactured and then integrated, for example, by applying an electric field to the support to some extent, the long carrier is bonded to the support only through one end in the longitudinal direction. It is possible to After that, the elongated carrier may be fixed to the support while maintaining that state.

For example, when a substrate made of a reactive substance such as a metal or a semiconductor is selected and carbon nanotubes are selected as the long and narrow carriers, they can be fixed as follows. That is, the substrate is heated to diffuse the reactant atoms into the carbon nanotubes. Next, it is possible to fix the carbon nanotubes to the substrate by partially modifying the carbon nanotubes to a carbide and forming a heterojunction between the reaction product obtained by the modification and the carbon nanotubes.

Alternatively, the substrate may be made of any substance, but when the carbon nanotubes are oriented vertically to the substrate, the carbon nanotubes are dispersed in a paste-like substance such as carbon paste. Is applied to a substrate and electrolysis is applied in the direction perpendicular to the substrate, whereby many carbon nanotubes are oriented in the direction perpendicular to the substrate. By drying the paste-like substance in this state, it is possible to obtain carbon nanotubes oriented in the direction perpendicular to the substrate. Also,
This method can also be used for elongated carriers made of substances other than carbon nanotubes.

The method for the substrate made of the above-mentioned reactive substance may be combined with the above-mentioned method using the paste. Thereby, it is possible to obtain a long and narrow carrier oriented in the direction perpendicular to the substrate. However, the method is not limited to this method, and the elongated carrier may be fixed to the support by a method known per se.

For example, when whiskers are used as the long and narrow carriers, a suitable substrate is selected, and crystals oriented on the substrate are grown by a method known per se, whereby desired whiskers extending to the surface of the substrate are obtained. May be synthesized and used as a long and narrow carrier. Further, the substrate used for forming such a long and thin simple body may be used as it is as the support of the present invention.

The size of the support used according to the present invention is not limited to this, but may be several mm to several cm.
May be It is sufficient that the long and thin carriers are arranged at a density of about 1 to about 5 × 10 ⁹ lines / cm ² with respect to the support having such a size. The higher the density of the immobilized nucleic acid probe, the higher the detection sensitivity and the better the S / N ratio.

According to the first aspect, it is preferable that the long and thin carriers are arranged on the surface of the support as an independent single body, without adjoining the long and thin carriers adjacent to each other. Therefore, it is preferable that the long and narrow carrier has hardness so as to enable such arrangement regardless of the material and shape.

Further, in the first mode, an example in which a plate-shaped support body is used as the support body has been described, but the shape is not limited to this, and a form including a sphere, a rotating body and a curved surface, etc. May be In that case, the elongated carrier may extend to at least a part of the surface of the support having such a configuration.

(4) Method of immobilizing the probe The immobilization of the probe on the solid phase surface may be performed by an immobilizing means known per se. For example, it is possible to immobilize the probe on the solid phase surface by using a means using a spotter or the like, a means using a general semiconductor technique, a means using a photocatalyst, or the like.

The immobilization of the probe on the elongated carrier may be carried out directly on the surface of the elongated carrier, or by subjecting the surface of the elongated carrier to a desired treatment, whereby chemical modification is carried out. Alternatively, it may be performed after physicochemical modification and / or physical modification.

The chemical modification, physicochemical modification and / or physical modification of the surface of the elongated carrier may be carried out by a method known per se generally used for immobilizing a probe on a support. Is possible. For example, such modification can be performed as follows.

(A) When the probe is immobilized by electrostatic binding For example, the surface of the elongated carrier may be treated with polylysine, polyethyleneimine, polyalkylamine or the like. By such a treatment, it is possible to apply the polycation to the surface of the elongated carrier. Thereby, if the substance having a negative charge such as nucleic acid or polypeptide is a probe, it is possible to electrostatically fix the probe by utilizing the negative charge.

(B) In case of fixing probe by coordination bond In addition, a film forming technique such as electron gun vapor deposition, sputtering, CVD, electrolytic plating and electroless plating may be applied to the elongated carrier. As a result, a receptor for coordination bond can be formed on the surface of the elongated carrier. The "coordination bond" here is a kind of chemical bond, and is a chemical bond in which an electron pair consisting of a donor and an acceptor and involved in the bond between atoms is donated from only one atom. is there. When a coordinative bond acceptor is formed on the surface of the long and narrow carrier, a chemical species that can serve as a coordinative bond donor is also bound to the probe. Thereby, it is possible to immobilize the desired probe on the elongated carrier by their coordinate bond. For example, examples of the combination of a coordinate bond acceptor and a donor are gold and sulfur, Z
n and RX (X is a halogen, R is a hydrocarbon) and the like, but are not limited thereto, and any combination may be used as long as it is a combination capable of obtaining a coordinate bond. Further, when the long carrier is formed of a substance that itself functions as a receptor, it is not necessary to form a film on the long carrier, and a coordinate bond is formed by introducing a desired functional group on the probe side. It is possible to achieve.

As described above, in the case where an acceptor or a donor of a coordinate bond is attached to the long and narrow carrier by film formation, the probe to be immobilized thereto forms a pair with it. A chemical species capable of being such a donor or an acceptor is provided. The means for giving the desired chemical species to the probe may be any means known per se.
For example, when a nucleic acid is selected as the probe, an enzymatic reaction may be used or a nucleic acid synthesizer may be used. The chemical species may also be introduced at any part of the nucleic acid, and may be introduced at the 3'end, the 5'end or at a random position.

(C) In case of immobilizing probe by covalent bond It is also possible to introduce a functional group into the elongated carrier.
By introducing a functional group into the long and narrow carrier, it is possible to bind a desired probe to the long and thin carrier by covalent bond.

Immobilization by covalent bonding includes, for example,
A method of activating the carrier surface and then directly or indirectly immobilizing the probe through a cross-linking agent, in which an active functional group is introduced into the probe to be immobilized on the elongated carrier to directly attach it to the elongated carrier. Alternatively, a method of indirectly fixing may be used. Here, the activation of the carrier surface can be performed, for example, by electrolytic oxidation in an oxidant, air oxidation, reagent oxidation, or coating with a film. Examples of the cross-linking agent that can be used include, but are not limited to, cyanogen bromide, silane couplers such as gamma-aminopropyltriethoxysilane, carbodiimide, and thionyl chloride.

On the other hand, when the functional group is introduced into the long and narrow carrier, it is preferable to introduce the functional group into the end of the probe to be immobilized therein. Examples of the functional group introduced into the probe include an amino group, a carboxyl group, a hydroxyl group, a carbonyl group, a phosphoric acid group, an aldehyde group, and a mercapto group,
The functional group is not limited to these, and other highly reactive functional groups may be used.

When the surface of the elongated carrier is oxidized to activate it, an oxide layer is formed there. The nucleic acid probe is bound to the elongated carrier through this oxidation layer. At this time, by reducing the thickness of the oxide layer, the S / N ratio in the case of electrochemically detecting a gene can be improved. The thickness of the oxide layer is preferably 500 Å or less, more preferably 100 Å or less.

When a nucleic acid is selected as the probe, the functional group can be introduced at the terminal of the nucleic acid by using an enzymatic reaction or a nucleic acid synthesizer. Examples of the enzyme used in the enzymatic reaction include terminal deoxynucleotidyl transferase, poly A polymerase, polynucleotide kinase, DNA polymerase.
, A polynucleotide adenyltransferase, R
NA ligase can be mentioned. Also, the polymer
Functional groups can also be introduced by the ze-chain reaction (hereinafter referred to as PCR), nick translation, or random primer method. The functional group may be introduced into any part of the nucleic acid and can be introduced into the 3 ′ end, the 5 ′ end or a random position.

The nucleic acid probe into which the functional group has been introduced can be directly immobilized on a carrier by an immobilization reaction. However, since the nucleic acid probe is a single-stranded nucleic acid, the amino group constituting the nucleic acid may function as the functional group, not the introduced functional group. That is, the probe is fixed on the carrier by the amino group constituting the nucleic acid. This causes a decrease in sensitivity and is not preferable.

The immobilization of the probe on the elongated carrier by the amino group constituting the nucleic acid probe can be prevented by, for example, the following method. First, the functional group-introduced nucleic acid probe is annealed with a nucleic acid chain having a sequence complementary to the probe to form a double-stranded chain. Next, the double-stranded nucleic acid is immobilized on a carrier by the introduced functional group, and then it is converted into a single-stranded nucleic acid by heat denaturation to remove the nucleic acid strand having no introduced functional group. The heating temperature at the time of heat denaturation is usually 90 to 98 ° C.

FIG. 2 shows examples of functional groups introduced into the support. 2A is a human loxyl group, B is a ketone group, C is a carboxyl group, E is an aldehyde group, and F is a thiol group.

When the probe is immobilized on the elongated carrier by covalent bond, the functional groups introduced into the elongated carrier and the probe are combined with each other to form a covalent bond. The functional group having reactivity may be selected.

Further, when carbon nanotubes are selected as the long and thin carriers, it is possible to open the ends of the carbon nanotubes by, for example, oxidation to form open carbon nanotubes. A carboxyl group is introduced into. Further, by heating in air using a means known per se, it is also possible to impart an —OH group, a —COOH group, a carbonyl group, a lactone ring, a quinone or a phenolic hydroxyl group.

When a long carrier is formed by selecting a material such as hydroapatite or polyoxymethylene, since they generally have a functional group, the probe is covalently bonded by using these functional groups. Can be fixed.

By further chemically modifying any of the functional groups, the desired probe can be covalently bonded. For example, in the case of an —OH group, it is possible to covalently bond a probe having a functional group necessary for covalent bonding to a long carrier by reaction with a Si crosslinking agent such as a silicifying agent. SOCl in case of carboxyl group
After passing through the -COCl via _2, by substitution reaction with the introduced probe NH ₂ group (hight carrier) -CO
Immobilization is achieved by covalent bonding in the form of NH- (probe). Regarding carbon nanotubes,
It is possible to utilize the known means of chemical modification used for graphite.

(D) In the case of immobilizing the probe by physical adsorption When the elongated carrier to which the nucleic acid probe is to be immobilized is an electrode, the physical adsorption enables efficient and simple operation of the nucleic acid probe. Can be fixed. Physical adsorption of the nucleic acid probe on the electrode surface can be performed, for example, as follows. First, the electrode surface is washed with distilled water and alcohol using an ultrasonic cleaner. Thereafter, the electrode was used as a phosphate buffer solution (pH 7.0) containing a nucleic acid probe.
And the nucleic acid probe is adsorbed on the surface of the carrier. At this time, 0 to +1.0 V, preferably 0 to +0.1 V to the electrode
By applying a potential within the range, adsorption of the nucleic acid probe can be promoted. Next, the electrode on which the nucleic acid probe was adsorbed was treated with nucleotides (ATP, CTP, GTP,
(TTP, dATP, dCTP, dGTP, dTTP, etc.)
The electrode surface is coated with nucleotides while being inserted into a solution and applying an electric potential preferably in the range of 0 to + 1.0V. This suppresses nonspecific adsorption of the sample nucleic acid, double-stranded chain-recognizing substance, etc., on the electrode surface. In addition, non-specific adsorption can be suppressed by surfactants, fatty acids, fats and the like.

(D) Immobilization of probe by other means The nucleic acid probe can also be immobilized on an elongated carrier by using an entrapping agent used in an encapsulation method known as one method of enzyme immobilization. The entrapping agent that can be used in the present invention is not particularly limited, but examples thereof include polyvinyl chloride and polyacrylamide.

The nucleic acid probe can also be immobilized on the electrode surface through a membrane. Examples of the film used in this case include conductive polymers such as polyacetylene, polypyrrole, polythiophene and polyaniline, polyethylene, polypropylene, polyvinyl chloride, polyvinyl alcohol, polymethylmethacrylate, polyvinylidene fluoride, Cellulose and lipid membranes can be mentioned. Alternatively, a monomolecular film such as an LB film or a film in which a plurality of monomolecular films are stacked to form a multilayer can be used.
The immobilization of the nucleic acid probe on the membrane can be carried out by the same method as the immobilization of the probe directly on the surface of the elongated carrier.

When the nucleic acid probe is immobilized on the membrane by covalent bonding, a functional group may be introduced into the membrane instead of introducing the functional group into the nucleic acid probe. As the functional group introduced into the membrane, the same functional group introduced into the nucleic acid probe can be used. By introducing a functional group into the membrane and then reacting and immobilizing the nucleic acid probe, the probe is immobilized at a higher density than when the functional group is introduced and immobilized in the nucleic acid probe. And a more stable nucleic acid probe-immobilized carrier can be obtained.

When the elongated carrier for immobilizing the nucleic acid probe through the membrane is an electrode, the probe is hybridized with the specimen sample as described above, and the membrane potential before and after the hybridization is performed. The presence or absence of the target gene can be detected by measuring the change.

The long and narrow carrier to which the nucleic acid probe is immobilized is likely to cause non-specific physical adsorption of the sample nucleic acid, the double-stranded chain-recognizing substance, etc. This causes a decrease in sensitivity. Such non-specific adsorption can be suppressed by immobilizing the nucleic acid probe and then coating the surface of the carrier with the nucleic acid by physical adsorption or chemical bonding.

In this case, examples of the nucleic acid for coating the surface of the carrier include nucleosides such as adenosine, thymidine, guanosine and cytidine, uridylic acid, cytidylic acid,
Nucleotides such as adenylic acid and guanylic acid, synthetic oligonucleotides, natural DNA such as salmon sperm DNA
Can be mentioned.

The length and sequence of the nucleic acid coating the surface of the carrier are not particularly limited as long as the length and sequence do not react with the nucleic acid probe immobilized on the surface of the carrier. A single-stranded or double-stranded nucleic acid having a length of -100 bases is desirable.

It is also possible to suppress non-specific adsorption by coating the elongated carrier with a substance such as a surfactant, fatty acid or fat. As such a substance, specifically, stearylamine or the like can be used.

According to the embodiment of the present invention, the immobilization of the nucleic acid probe on the elongated carrier provided on the probe-immobilized support is not limited to the above-mentioned method. The method used for immobilization on a solid phase can be widely used.

(6) Effect of the first aspect The probe fixing support having the structure according to the first aspect can increase the number of probes without increasing the size of the entire apparatus, thereby improving the sensitivity of detection and analysis. improves.

That is, the device according to the first aspect provides a device which is a small device and can detect a biological substance with higher sensitivity than before.

When a carrier having a long and thin structure such as a linear shape, a fibrous shape, or a columnar shape is used, the portion on which the probe is fixed becomes three-dimensional, and the specific surface area increases accordingly. As a result, many probes can be immobilized. Therefore, highly sensitive analysis can be performed in a smaller area than in the past.

Although the probe fixed support formed on one support is shown here, a plurality of probe fixed supports according to the first aspect having arbitrary sizes are prepared if desired. It may be further arranged on the second support.

Further, in the probe fixing support shown in the first embodiment, the example in which the probe is fixed only on the surface of the long and thin carrier has been shown, but this is not necessarily the case, and it is not necessary that the long and narrow carriers be adjacent to each other. The probe may be fixed to the surface of the support.

2. Second Aspect FIG. 3 is a drawing schematically showing the probe fixing support according to the second aspect. 3A is a side view of the probe fixing support, and FIG. 3B is a plan view thereof. As shown in FIG. 3A, the probe fixing support 31 includes a flat substrate 32 as a support, and a long thin strip extending on one surface of the substrate 32 through one end thereof and arranged in a bundle. A carrier 33 and a probe 34 fixed on the surface of the elongated carrier 33 are provided.

The second aspect is the same as the probe fixing support of the first aspect described above except that the elongated carriers are arranged in a bundled state. Therefore, any part can be changed in the same manner as in the first aspect. Further, the same effect as that of the first aspect can be obtained.

In the example shown in FIG. 3, the number of long and thin carriers per unit is four, but the number is not limited to this, and two or more long and thin carriers are arranged in a bundle. do it. Further, the first aspect and the second aspect may be simultaneously present on the same support.

Furthermore, the first probe fixing support is provided with a first
The immobilization mode of the elongated carrier according to the second aspect and the immobilization mode of the elongated carrier according to the second aspect may be mixed.

3. Third Mode (1) Third Mode A third mode is shown in FIG. FIG. 4 is a drawing schematically showing the probe fixing support according to the third aspect. Figure 4A
4B is a side view of the probe fixing support, and FIG. 4B is a plan view thereof. FIG. 4C is an enlarged view of only the portion on which the probe solution is spotted in the probe fixed support shown in the plan view of FIG. 4B (the spotted portion is within the circle of FIG. 4B). As shown in FIG. 4A, the probe fixing support 36 is
It comprises a flat substrate 37 as a support, an elongated carrier 38 extending through one end of the surface of the substrate 37, and a probe 39 fixed to the surface of the elongated carrier 38.

Here, the distance between adjacent elongated carriers 38 is shorter than the length of the probe fixed thereto.
That is, when d is the distance between the elongated carriers and D is the length of the probe, the equation of d <D is established.

When d <D, as can be seen from FIGS. 4 (A), (B) and (C), one probe 39 is provided for each elongated carrier 38 in the spotted portion. Is fixed. This is because a plurality of probes 39 cannot enter there because the distance between the elongated carriers is small. The elongated carriers 38 arranged in this way are considered to exist as an aggregate that receives the influence of the adjacent elongated carriers 38.

By having such a configuration, the third
In this mode, it is possible to make the density of the probe fixed per target area of the probe fixed support constant.

The size of the support used according to the present invention is not limited to this, but it is from several mm to several cm.
May be It suffices that the long and thin carriers are arranged at a density of about 1 × 10 ^{11 to} 5 × 10 ¹³ lines / cm ² on the support having such a size.

(2) Comparison of First and Third Aspects As described above, the difference between the first and third aspects is that the elongated carriers are arranged as a single body on the support or are aggregated. It is arranged as a body. Specifically, in the case of the probe fixing support of the first aspect, as shown in FIGS. 5A and 5B, each elongated carrier 41 is provided between adjacent elongated carriers 41.
There is a distance (Fig. 5A) (d> D) where the probes 42 included in the probe do not come into contact with each other, or a distance longer than the total length of the probes fixed to the adjacent elongated carriers (Fig. 5B) (d).
≧ D; where d is the distance between the elongated carriers and D is the length of the probe). On the other hand, in the third aspect, adjacent long carriers are in contact with each other, or there is a distance shorter than the entire length of the probe fixed thereto (d <D) (FIG. 6).

Here, FIG. 6A is an enlarged view of a portion of the probe fixing support according to the third embodiment on which the probe solution is spotted. Only in the spot where the probe solution is spotted, the probe has about 1: 1 probe-to-long carrier, several pairs to 1 or 10: 1 to the tip of the long carrier to measure or detect the target substance. It is fixed to the extent that it can maintain a certain degree of density consistency necessary for.

The third embodiment shown here can be manufactured in the same manner as the first embodiment except that the elongated carriers are arranged as an aggregate, and such an elongated carrier can be manufactured. Except for the arrangement, it can be arbitrarily changed according to the first aspect.

4. Fourth Mode As a fourth mode, an example in which carbon nanotubes are used as elongated carriers will be described. The probe fixing support according to the fourth aspect has the same configuration as that of the first aspect shown in FIG. 1 except that carbon nanotubes are used as the elongated carriers.

The probe-immobilized support of this embodiment is, for example,
It can be manufactured as follows. Next, on a suitable substrate such as a silicon substrate (hereinafter referred to as Si substrate),
When a hydrocarbon catalytic decomposition method in which a transition metal catalyst particle such as iron or cobalt is reacted with a hydrocarbon such as acetylene at a high temperature, carbon nanotubes oriented on the substrate are formed. Further, a probe may be fixed to this carbon nanotube.

For example, when a nucleic acid is used as a probe to be immobilized on the elongated carrier of the present invention, carbon nanotubes having a diameter of several nm and an outer shape of linear, fibrous or columnar shape are , Which is referred to as a nucleic acid probe), has a significant radius of curvature with respect to the length thereof, so that the nucleic acid probe is radially fixed to the carbon nanotube. That is, the probes are radially fixed along the radiation extending outward from the center of the cross section obtained when the carbon nanotubes are cut in a plane parallel to the substrate surface. In such radial fixation, the distance between adjacent nucleic acid probes becomes larger than that in the case where the probes are fixed so as to directly extend vertically on a flat plate. Therefore, even if the nucleic acid probe is immobilized on the carbon nanotube with the same binding density as that of the conventional DNA chip in which the probe is directly immobilized on the surface of the substrate, there is a possibility that unexpected coupling between the probes will occur. Is also significantly reduced. As a result, the reaction efficiency of hybridization and PCR is increased, and the reliability of detection and quantitative analysis is improved.

DNA fixed on a plane as in the past
When the target DNA (hereinafter referred to as the target DNA) is hybridized with the probe, it is considered that the density is preferably 10 ¹¹ to 10 ¹³ lines / cm ² or less. The target DNA cannot enter between the DNA probes that are more closely packed, and the hybridization that should occur is inhibited. Therefore, conventional DN
With the A chip, it was difficult to achieve a high density of 10 ¹¹ to 10 ¹³ lines / cm ² or more. However, when the DNA probe is immobilized on the carbon nanotube, the DNA probe is radially bonded to the carbon nanotube as described above, and therefore the density of the immobilized DNA probe is 10 ¹¹ to 10 ¹³ / cm ^2. Even if the density becomes higher than the above, a sufficient gap can be formed between the probes. Therefore,
Hybridization is efficiently achieved even at high density.

The carbon nanotube has a diameter of several n.
While the length is m, the length is about several μm. That is, 10
Since it has a high aspect ratio of 00 times or more, the specific surface area can be easily increased, and the density of probe fixation can be increased.

In this embodiment, the structure may be changed according to the first embodiment, except that carbon nanotubes are used as the long and narrow carriers to be used.

5. Fifth Mode As a fifth mode, an example in which carbon nanotubes are used as elongated carriers will be described with reference to FIG. 7. The probe fixing support according to the fifth aspect has the same configuration as that of the third aspect shown in FIG. 4, except that carbon nanotubes are used as the long and narrow carriers and adjacent long and narrow carriers are in close contact with each other. . For the sake of convenience, FIG. 7 shows nine elongated carriers that are present on the inside shown in FIG. 4 (FIGS. 7A and 7B).

FIG. 7A is a perspective view schematically showing a probe fixing support 60 according to the fifth mode. The probe fixing support 60 is a device in which carbon nanotubes 62 as long and thin carriers are arranged on the surface of a substrate 61 as a support so as to be in contact with each other, and a probe is fixed on the surface of the carbon nanotubes 62.

In order to dispose the elongated carrier on the substrate with such a structure, for example, Si having a Si surface and a C surface is used.
The carbon nanotubes may be synthesized by heating the C surface of the C substrate in vacuum. Then, a probe is fixed on the surface of the carbon nanotube.

Further, even if the method other than the above-mentioned example is used, it is possible to manufacture the carbon nanotube extending to the substrate by a method known per se. For example, fine particles of the metal catalyst are arranged in desired pores of zeolite having uniform pores, and the metal catalyst fine particles are used as nuclei for chemical vapor deposition (that is,
By performing the CVD method), it is possible to extend the carbon nanotubes as a long and narrow carrier on the porous substrate. The zeolite used in this case is iron acetate,
It is possible to manufacture by mixing cobalt acetate and zeolite in an aqueous solution and heating. When manufactured in this way, the cobalt and iron catalyst particles are arranged in the pores of the zeolite. Using this, carbon nanotubes may be grown by the CVD method.

The probe fixing support 60 according to this embodiment is
The distance between the adjacent carbon nanotubes 62 is very small. Therefore, when the probe is fixed to the carbon nanotube 62, it is difficult for the probe to diffuse to the side wall of the carbon nanotube 62. As a result, the probe 6
3 binds only near the tip of the carbon nanotube (FIG. 7A).

The tendency of the probe to bind to the carbon nanotube 62 provided on the substrate 61 is schematically shown in FIG. 7B. FIG. 7B is a plan view of the substrate including the carbon nanotubes before the probe is fixed. For example, when a nucleic acid as a probe is immobilized on a carbon nanotube arranged on a substrate, the binding of the nucleic acid probe thereto occurs under three different probabilities depending on the site. FIG. 7B shows the distribution of such three-staged joint probabilities. Carbon nanotube 62 indicated by a black circle
The central portion B1 at the tip of is a site where binding occurs with high probability.
The B2 region shown in white is a site capable of binding, although it is inferior to B1. The B2 region is a portion of the side surface of the carbon nanotube other than the portion closest to (or in contact with) the adjacent carbon nanotubes, that is, a portion having a taper from the side surface of the carbon nanotube toward the tip. Further, the shaded B3 region is the exposed portion of the substrate, and this portion is a portion that is difficult to bond.

As described above, due to the difference in the degree of probe binding depending on the region, in the probe-immobilized support according to the fifth aspect, the immobilization density (ie,
The existing density depends on the density of the carbon nanotubes existing on the substrate. As a result, when the nucleic acid probe is fixed by spotting, the nucleic acid probe can be fixed at a constant density without depending on the density of the nucleic acid probes contained in the droplets of the probe solution to be dropped. Therefore, reliability in quantitative analysis is improved.

Here, the reason why one nucleic acid probe is fixed per carbon nanotube is due to the balance of the thickness of the carbon nanotube and the nucleic acid probe and the steric hindrance due to the nucleic acid probe. Therefore, due to such balance and steric hindrance, the density of the carbon nanotube and the probe is about 1: 1 or several to 1: 1 or about 10: 1, and the density is constant to some extent necessary for measuring or detecting the target substance. Similar effects can be obtained as long as immobilization can be achieved to the extent that the property can be maintained, that is, if the thickness is close to the thickness of the nucleic acid. Such substances are also preferably used as the probe. Examples of substances used as probes according to the fifth aspect are various nucleic acids and nucleic acid analogues, polypeptides and antibodies and the like.

When the probe is solid-phased on the carbon nanotube extending from the substrate 61 with such a structure,
The carbon nanotube may be chemically modified, physicochemically modified, and / or physically modified as described in the first embodiment, and then the solid phase of the target probe may be performed. Further, at the same time as such treatment of carbon nanotubes, modification such as introduction of a functional group may be performed on the probe. The practitioner may arbitrarily select which treatment is to be performed based on the type of probe used, the labeling substance, the detection method, and the like.

When carbon nanotubes are extended from the surface of the SiC substrate, the surface of the SiC substrate is 10 ¹¹
Carbon nanotubes are formed at a density of 10 ¹³ / cm ² . This density is the optimum density for carrying out a nucleic acid hybridization reaction. Therefore, by fixing one nucleic acid probe for each carbon nanotube of such a density, it is possible to fix the probes at a density of 10 ¹¹ to 10 ¹³ / cm ² , which is the optimum density for hybridization. is there. This improves the reaction efficiency of hybridization and PCR reaction.

Here, an example of the carbon nanotube is shown, but as long as such a constitution can be achieved, it may be manufactured from other materials, and may be arbitrarily changed according to the description in the first aspect.

6. Sixth Mode FIG. 8A shows a sixth mode in which the probe fixing support shown in the fifth mode is partially modified. The probe fixing support according to the sixth aspect comprises carbon nanotubes having an open tip as an elongated carrier. Hereinafter, description will be given with reference to FIG. 8A. In the probe fixing support 70, carbon nanotubes 72 as a plurality of long and narrow carriers are fixed to a base 71, and the ends of the carbon nanotubes 72 are opened.
The opening 74 of the opened carbon nanotube 72
The probe 73 is fixed to the edge 75 of the.

As the means for opening the tip of the carbon nanotube 72, it is possible to utilize means known per se such as oxidation, etching and laser ablation, and reflux in nitric acid or hydrogen peroxide. . Further, by heating in air using a means known per se, it is also possible to impart an —OH group, a —COOH group, a carbonyl group, a lactone ring, a quinone or a phenolic hydroxyl group.

Next, please refer to FIG. 8B. FIG. 8B is a plan view of the substrate including the carbon nanotubes before the probe is fixed. By opening the tip of the carbon nanotube 72, the carbon portion forming the carbon nanotube 72, the opening portion 74 of the carbon nanotube, the peripheral carbon portion B1 framing the opening portion 74, and the carbon portion B1 are formed on the substrate 71. There is an inner cavity B2 defined by the inside and an exposed substrate surface B3. Fifth
Similar to the above embodiment, the binding of the probe to each site of B1, B2 and B3 is biased. That is, the peripheral carbon portion B1 is the portion to which the probe is most likely to bind, the exposed substrate surface B3 is the portion to which the probe is difficult to bind, and the inner cavity portion extending in the direction from the opening 74 of the carbon nanotube toward the substrate. B2 depends on the size of the inner diameter of the hole, but is generally a region where probe coupling is extremely difficult. The density of probes present in the device depends on the density of carbon nanotubes.

In the structure of the probe fixing support according to the present invention as shown in the sixth aspect, the area of the solid phase surface is limited by the openings of the densely packed carbon nanotubes.
Thereby, the probe binding density can be made constant without depending on the dispersion of the dropped probe solution. This improves the reliability when performing quantitative analysis.

The sixth mode is the same as the fifth mode except that the carbon nanotubes are open-ended. Therefore, the sixth aspect may be modified as desired in the same manner as the fifth aspect described above.

Further, according to the present invention, as a modification of the sixth aspect, there is provided a probe fixing support in which the inner diameter of the carbon nanotube is sufficiently increased. If the carbon nanotube is manufactured so as to have the same configuration as that of the sixth embodiment except that the inner diameter of the carbon nanotube is made sufficiently large, one or more probes are fixed to B2 of the inner cavity of the carbon nanotube according to the size of the inner diameter. Is possible.

7. Seventh Mode In the fifth mode and the sixth mode, an example in which the carbon nanotubes 62 or 72, which are adjacent to each other and extend on the substrate 61 or 71 so as to extend in close contact with each other, is used as the elongated carrier. Particularly, in the sixth aspect, as a whole, such carbon nanotubes exist as a film formed on the substrate. Also, if adjacent long and narrow carriers come close to each other,
Adjacent elongated carriers share one wall surface. Therefore, in view of the present invention from such a viewpoint, the aggregate of the long and narrow carriers can be taken as a porous body in which the pore sizes are almost the same. Therefore, in the present invention, it is also possible to manufacture the probe-immobilized support of the present invention by using a porous material having pores of substantially the same size as an aggregate of long and narrow carriers.

The porous member preferably used according to the present invention may be, for example, a mesoporous substance known per se. Examples of mesoporous materials are inorganic layered crystals of layered silicates, also called mesoporous silica, such as KSW-2 and FSM-16, and inorganic organic layered hybrids composed of silicon alkoxides.
However, the present invention is not limited to this, and any member can be used as long as the member has holes regularly and uniformly in a few nanometers.

According to an aspect of the present invention, it is desirable that the inner diameter of the hole be shorter than the length of the probe. When the inner diameter of the porous hole is shorter than the total length of the probe fixed thereto, the probe is unlikely to enter the inside of the hole when fixing the probe. Therefore, the probe is fixed to the edge of the hole as in the sixth aspect. That is, since the area of the solid phase surface is limited by the existence of the holes, the binding density of the probe can be made constant without depending on the dispersion of the dropped probe solution. This improves the reliability when performing quantitative analysis.

[0126] In a further aspect of the present invention, there is provided a probe immobilizing support comprising a porous body having pores of approximately the same diameter and a probe fixed to the edge of the porous pores.
Such a probe fixed support is also included in the scope of the present invention.

II. Detection mode The detection of a target substance using the probe-immobilized support of the present invention is carried out by binding the probe immobilized on the support to the target substance in the sample in the following manner: (1) a method using a labeling substance or (2) It can be detected by any means of electrochemical methods.

For example, when the probe is a nucleic acid, both the methods (1) and (2) can be used. Hereinafter, the detection method will be described by taking the case where the probe is a nucleic acid as an example.

In the case of the method using the labeling substance of (1), the sample nucleic acid is FITC, Cy3, Cy5, rhodamine, N-acetoxy-N ₂ -acetylaminofluorene (hereinafter referred to as AAF) or AAIF (ie, AAF (iodine derivative of AAF) or the like, or biotin, hapten,
It is labeled with an enzyme such as oxidase or phosphatase, or an electrochemically active substance such as ferrocene or quinones. Alternatively, detection is performed by using a second probe labeled with the above-mentioned substance. Multiple labeling substances can be used simultaneously.

In the case of the electrochemical method (2), a conductive substance may be used as the elongated carrier for immobilizing the nucleic acid probe, and the probe-immobilized support may be used as an electrode. You may give it a function and use it. The detection of the hybridization reaction using this electrode may use a counter electrode or a reference electrode in addition to this electrode, as in other general electrochemical detection methods. When disposing the reference electrode, a general reference electrode such as a silver / silver chloride electrode or a mercury / mercury chloride electrode can be used. In order to arrange the reference electrode on the probe-immobilized support according to the present invention, for example, when the carbon nanotube shown in the third aspect is used as an elongated carrier on a Si substrate, the reference electrode is arranged on the Si substrate. You may arrange. Further, the reference electrode and / or the counter electrode need not necessarily be arranged on the substrate, and a means that can be present in the reaction solution may be used. Further, the reference electrode does not necessarily have to be arranged.

The elongated carrier used in the present invention is not limited to these in order to have a function as an electrode, but usable examples thereof include graphite, glassy carbon, pyrolytic graphite and carbon. −
Carbon electrodes such as carbon fiber and carbon nanotubes, platinum, platinum black, gold, precious metal electrodes such as palladium and rhodium, oxide electrodes such as titanium oxide, tin oxide, manganese oxide and lead oxide, Si, Ge, ZnO,
Examples thereof include semiconductor electrodes such as CdS, TiO ₂ and GaAs, titanium and the like. These electrodes may be coated with a conductive polymer, whereby a stable probe-immobilized electrode can be prepared. It can also be covered with a monolayer. The nucleic acid probe can be immobilized on the elongated carrier which is the surface of the electrode by covalent bond, ionic bond, physical adsorption or the like.

Further, the probes having different base sequences may be separately immobilized on the conductive elongated carrier provided on one support for each type of probe, and all the probes provided on one support may be fixed. The same probe may be fixed to the conductive elongated carrier, and a plurality of such probes may be arranged on a support such as another substrate.

[0133] In some embodiments, the hybridization reaction between the nucleic acid component extracted from the sample substance and the nucleic acid probe immobilized on the probe-immobilized support is carried out by, for example,
It would be done as follows. The hybridization reaction solution has a pH of 5 at an ionic strength of 0.01 to 5.
It is carried out in a buffer solution in the range of -10. Dextran sulfate, which is a hybridization promoter, salmon sperm DNA, calf thymus DNA, EDTA and a surfactant may be added to this solution. The extracted nucleic acid component is added thereto, and heat denaturation is performed at 90 ° C or higher. Insertion of the probe-immobilized support may be performed immediately after denaturation or after rapid cooling to 0 ° C. Alternatively, the hybridization reaction may be performed by dropping a liquid on the probe-immobilized support. During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature is, for example, in the range of 10 ° C to 90 ° C,
The reaction time may be 1 minute or more and about 1 night. After the hybridization reaction, the electrode is taken out and washed. For washing, for example, in the range of ionic strength 0.01 to 5, pH
Buffers in the range 5-10 are used.

In the case of the method using the labeling substance of (1), the hybridization reaction is detected by using an appropriate detection device according to the type of the label, in which the labeled base sequence in the sample or the secondary probe It may be performed by detecting the labeling substance. When the labeling substance is a fluorescent substance, for example, a fluorescence signal may be detected using a fluorescence detector.

In the case of the electrochemical method (2), detection is carried out by the following procedure. First, after reacting a sample nucleic acid with a nucleic acid probe on a probe-immobilized support (hereinafter also referred to as a support), the support is washed. After that, a double-stranded chain-recognizing substance that selectively binds to the double-stranded chain formed on the surface of the long carrier as an electrode is made to act, and the signal from the double-stranded chain-recognizing body bound there is measured electrochemically do it.

The double-stranded chain-recognizing agent used here is not limited to these, but is, for example, Hoechst 332.
58, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, tris intercalator, polyintercalator and the like can be used. Further, these intercalators are electrochemically active metal complexes,
For example, it may be modified with ferrocene, viologen, or the like.

The concentration of the double-stranded chain-recognizing substance varies depending on its type, but it may generally be in the range of 1 ng / mL to 1 mg / mL. At this time, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 may be used.

In the electrochemical measurement, a potential higher than the potential at which the double-stranded chain-recognizing body reacts electrochemically is applied, and the reaction current value derived from the double-stranded chain-recognizing body is measured. At this time, the potential is generated by sweeping at a constant speed, applying a pulse, or applying a constant potential. In the measurement, the current and voltage are controlled by using a device such as a potentiostat, a digital multimeter and a function generator. Based on the obtained current value,
The concentration of the target nucleic acid can be calculated from the calibration curve.

When the gene is detected by using the electrode, an intercalating agent which produces electrochemiluminescence can be used. Such an intercalating agent is not particularly limited, and examples thereof include luminol, lucigenin, pyrene, diphenylanthracene and rubrene. Electrochemiluminescence with these intercalating agents can be achieved by using luciferin derivatives such as firefly luciferin and dehydroluciferin, phenols such as phenylphenol and chlorophenol, or enhancers such as naphthols. It is possible to increase.

The electrochemical method does not require labeling of the sample nucleic acid. Further, the detection is performed by measuring an electric signal. Therefore, complicated systems such as those required for fluorescence detection are not needed. Therefore, such a method can be expected to reduce the size of the system.

III. Example of use The following is an example of use of the probe-immobilized support according to the present invention.

1. Analysis of Nucleic Acid Detection of nucleic acid contained in a specimen sample using the probe-immobilized support according to the present invention can be performed as follows.

First, a sample nucleic acid to be analyzed is prepared. The sample nucleic acid is, for example, a method known per se from a sample containing nucleic acid such as blood such as peripheral venous blood collected from an individual, white blood cells, serum, urine, feces, semen, saliva, cultured cells, organs and tissues. It may be extracted and prepared by. The term "individual" as used herein may be humans, non-human animals and plants, and microorganisms such as viruses, bacteria, bacteria, yeasts and mycoplasmas.

If desired, prior to the extraction of nucleic acids, cell disruption or tissue homogenization may be performed. When the amount of nucleic acid extracted from the sample is very small, it may be amplified by an amplification means such as PCR.

The sample nucleic acid thus prepared is added to the surface of the probe-immobilized support according to the present invention, which comprises the nucleic acid probe containing the desired sequence. Subsequently, hybridization may be carried out under appropriate conditions, for example, stringent conditions. As for the detailed reaction conditions such as temperature and reaction time selected in the hybridization, general conditions known per se can be used, but the selection depends on the type of base sequence to be detected, the used It may be arbitrarily selected by the practitioner according to conditions such as the type of probe nucleic acid to be used.

After the hybridization, the presence of the sample nucleic acid hybridized with the probe provided on the probe-immobilized support is detected. For this detection, for example, a sample nucleic acid is labeled with a labeling substance in advance,
This can be done by detecting the signal from the labeling substance. Alternatively, sandwich hybridization may be performed on the sample nucleic acid without labeling. Alternatively, a double-stranded nucleic acid generated by hybridization of a probe provided on a probe-immobilized support and a sample nucleic acid may be electrochemically detected using a double-stranded recognition body. In addition, as described above, various detection methods can be used depending on the configuration of the probe fixed support.

By such an operation, it is possible to analyze the base sequence of the sample nucleic acid. In addition, by selecting a probe to be immobilized on the probe-immobilized support, it can be used for various gene analysis and gene diagnosis. For example, detection and quantification of the presence of the target sequence, expression analysis to analyze the appearance and disappearance of gene expression, single nucleotide polymorphism in the genome (Single Nucleotide Polymorphism, ie,
SNP) and microsatellite sequence polymorphism analysis, disease-related gene analysis to diagnose disease and predict risk of onset, individual constitution, drug treatment effect analysis, detection of the presence of infection, detection of infection It can be used when conducting analysis of existing virus types and conducting toxicity tests. Further, it may be used to analyze the function of the genome in various organisms, and may be used in various clinical analyzes such as clinical diagnosis and onset prediction. Also, for example, food inspection,
It can be widely used in various basic researches and applied researches such as quarantine, drug inspection, forensic medicine, agriculture, livestock, fishery and forestry.

An analysis method using such a probe-immobilized support according to the present invention is also included in the scope of the present invention.

Further, the probe-immobilized support according to the present invention can be repeatedly used as a gene detection sensor for detecting a gene. For that purpose, after the measurement, the sample nucleic acid hybridized with the immobilized probe may be dissociated. The sample nucleic acid can be dissociated from the probe by heat treatment, alkali treatment, acid treatment, surfactant treatment, ultrasonic treatment, or the like.

For example, the heat treatment may be carried out by treating the sample at 98 ° C. for 5 minutes to denature the sample and then quenching. The alkaline treatment may be carried out by treating with a buffer solution having a pH of 8.5 or more or a strong alkaline solution. The acid treatment can be carried out by treating with a buffer solution having a pH of 4.5 or less or a strong acid solution. Further, the treatment with the surfactant is not limited to this, but for example, S
It can be performed using an ionic or neutral surfactant such as DS, Triton-X, and tween20. At this time, the concentration of the surfactant is preferably 0.1% or more. The ultrasonic treatment may be performed at a frequency of 10 KHz to 100 KHz for several seconds to several minutes.

2. Use of Probe-Immobilized Support in Nucleic Acid Amplification Nucleic acid extension and / or amplification may be performed on the probe-immobilized support according to the present invention. In that case, the nucleic acid used as the probe may be used as the template, or the sample nucleic acid bound to the probe may be used as the template.

When using the probe as a template,
The length of the probe may be any length as long as it is generally used as a probe. Further, as the primer used by binding to the template, both the forward primer and the reverse primer may utilize the target nucleic acid contained in the sample nucleic acid in the sample, or the target nucleic acid originally contained in the sample nucleic acid may be used as one primer. Then, the other primer may be added, or a known primer set (that is, a set of forward and reverse primers) may be added to carry out the amplification reaction.

When the target nucleic acid is used as a template, a probe having a sequence complementary to some base sequences at either end of the target nucleic acid is placed on the probe-immobilized support. The sample nucleic acid is hybridized with the probe. Then, the probe is caused to function as a primer, and nucleic acid extension and / or amplification reaction is performed using the sample nucleic acid as a template. Also, when performing the extension and / or amplification reaction,
Additional primers that pair with the probe may be used if desired. Alternatively, capture the target nucleic acid with a probe,
The extension and / or amplification reaction may be performed by adding a primer set.

As a method for amplifying nucleic acid, a polymer is used.
A method using an enzyme such as ze chain reaction (that is, PCR) is typical. However, the present invention is not limited to this, and any reaction may be used as long as it is extension and / or amplification using a primer and an enzyme.

Examples of the enzyme used in the gene amplification method include a DNA-dependent DNA polymerase such as DNA polymerase and Taq polymerase, and RNA.
DNA-dependent RNA Polymerase such as Polymerase I
And RNA-dependent RNA polymerases such as Qβ replicase.

The reaction conditions for carrying out such extension and / or amplification of nucleic acids may be appropriately selected by the practitioner depending on the sequences of the nucleic acids involved in the reaction, the enzyme and the like.

The present invention provides a method for extending and / or amplifying a nucleic acid on the probe-immobilized support according to the present invention. Further, a nucleic acid obtained by such a nucleic acid extension and / or amplification reaction is also within the scope of the present invention. Further, reagents and pharmaceuticals containing the nucleic acid obtained by such a method are also within the scope of the present invention. Further, a diagnostic method using the probe-immobilized support of the present invention, a method of predicting a disease incidence, a method of predicting a therapeutic effect of a drug, etc. are also within the scope of the present invention.

In such a probe-immobilized support according to the present invention, the probe is firmly bound to the elongated carrier. Therefore, when the probe is used as a template, it is possible to repeatedly subject the probe-immobilized support to the reaction by removing the nucleic acid other than the probe after performing the desired reaction. In that case, the sample nucleic acid may be dissociated as described in "1. Analysis of nucleic acid" in the preceding section.

3. Use as storage means The probe-immobilized support according to the present invention comprises a ribonucleic acid (RN).
A), deoxyribonucleic acid (DNA), peptide nucleic acid (P
NA), methylphosphonate nucleic acid, oligonucleotides such as S-oligo, nucleic acids and nucleic acid analogs such as polynucleotides such as cDNA and cRNA, peptides, polypeptides and proteins, and sugar chains, enzymes, antigens and antibodies It may be used as a means for transporting and / or storing bio-related substances such as bio-related substances and their analogs.

When the probe-immobilized support according to the present invention is used as a means for carrying and / or storing, a method similar to that for immobilizing the probe after purifying the biological substance to be stored. It may be fixed on the surface of the elongated carrier. Then, it may be stored in a refrigerator or a freezer.

Alternatively, a probe capable of specifically binding to a biological substance to be preserved is immobilized on a long carrier provided on a support, and the biological substance to be preserved is bound to the probe. It may be stored in a refrigerator or a freezer. In this case, after the storage for a desired period of time, when the biological substance is used again, it may be dissociated from the probe.

A method of using such a probe-fixed support as a transportation means and / or a storage means is also included in the scope of the present invention.

When the present invention is used, since the biologically relevant substance is immobilized on the surface of the solid-phase carrier in a purified state, no special storage facility is required, and samples can be stored easily and in large quantities for a long period of time. Is. The storage can also be performed in a home refrigerator. Therefore, handling of a sample containing a nucleic acid such as a gene sample becomes much easier and more convenient than in the past.

Conventionally, nucleic acids such as DNA and polynucleotide and samples containing nucleic acids have been frozen and stored at −80 ° C. in a solution state, immobilized on a membrane, or introduced into a host cell. For example, storage of DNA in a host cell state requires complicated steps, such as the need to gradually lower the temperature, and requires time and labor. In addition, conventional frozen storage requires a large storage space, which is a problem. Such a conventional problem is solved by the probe fixing support of the present invention.

IV. Probe-immobilized carrier Further, the elongated carrier according to the present invention as described above can be used as a probe-immobilized carrier in a state where the probe is immobilized, independently of the probe-immobilized support. The probe solid phase carrier according to the present invention is a very fine carrier as described above. By using such a fine carrier, it is possible to carry out a test with a smaller volume. For example, when the probe solid-phase carrier according to the present invention is used, it may be used by holding it in a solution contained in a reaction container such as a microtube, a test tube and a microtiter plate, instead of being provided on a support. It is possible.

[0166] That is, as a further aspect, the present invention provides a probe-immobilized carrier comprising an elongated carrier and a plurality of probes immobilized on the elongated carrier and specifically binding to a target substance.

The elongated carrier used according to this aspect may be any elongated carrier described in the first to fifth aspects.

In particular, if carbon nanotubes are used as the long and thin carriers, it is possible to control the number of probes fixed on the carbon nanotubes. For example, the carbon nanotubes may be removed from the substrate after fixing the probe to the carbon nanotubes formed on the SiC substrate. Moreover, since the probes fixed to the carbon nanotubes are fixed radially, it is possible to efficiently carry out the reaction between the probes and the target substance.

The present invention will be further described below with reference to examples.
These examples are provided by way of illustration and not limitation of the invention.

Examples Example 1. A probe-immobilized support including carbon nanotubes, which are columnar inorganic crystals of carbon nanotubes grown from a Si substrate, is manufactured.

(1) Production of long and narrow carrier First, 10% of didodecyl ammonium bromide was dissolved in toluene in a glove box sufficiently replaced with N ₂ . CoCl ₂ .6H ₂ O was added to this solution 0.005
M Dissolve and stir. Then, add 5M NaBH ₄ and mix and stir again. By this process, Co
Nanoparticles are dispersed in the solvent. The Co nanoparticles are washed with acetone and toluene, and finally Co nanoparticles are dispersed in acetone. A Si substrate is dipped in this solution and dried at room temperature. As a result, Co nanoparticles of about 5 nm are dispersed on the Si substrate at about 50 particles / μm ² .

FIG. 9a shows a state in which the particles are dispersed in the density.
It is shown as a formula. As shown in FIG. 9a, the Ni fine particles 81
It is dispersed on the surface of the Si substrate 82. At this time, the Si substrate surface
Using a scanning electron microscope (hereinafter referred to as SEM)
FIG. 10A shows a schematic diagram of the result of the observation. Then this
After introducing the Si substrate 82 into the thermal CVD chamber, _Two
H diluted with_TwoS + H_Two2 hours before at 400 ° C in gas
To process. Next, after decompressing the inside of the chamber, N
_TwoReplace with N_TwoDiluted C_TwoH_TwoFlow gas, 600
Pyrolysis is performed at ~ 1000 ° C to form a carbon film (Fig. 9).
b). The thermal CVD chamber used here has a mass
A flow rate controller (hereinafter, referred to as MFC) is provided.
This M. F. N through C_Two, H_TwoS, H_TwoAnd C_TwoH
_TwoAre appropriately introduced (FIG. 9b). Also, the thermal CVD chamber
The bar is equipped with a rotary pump (denoted as RP in the figure).
Be equipped.

By this step, carbon nanotubes 83 having a diameter of 30 to 80 nm, a length of 5 μm, and a density of about 10 pieces / μm ² are grown perpendicularly to the substrate by using Co nanoparticles dispersed on the Si substrate 82 as nuclei. (Fig. 9c).
The distance between adjacent carbon nanotubes is 100 n.
It is about m. This distance is longer than the length of a commonly used nucleic acid probe. Therefore, each of the carbon nanotubes functions as an independent long and thin carrier. Further, the Si substrate 82 used here is used as a support according to the present invention. Figure 1 shows a schematic diagram of the results of SEM observation of the created carbon nanotubes.
Shown in 0B.

In this example, an example in which carbon nanotubes were grown from a Si substrate was shown, but any substrate can be used as long as it is a heat resistant substrate such as zeolite, SiC substrate, glass substrate and α-Al ₂ O ₃ substrate. May be used. It is possible to obtain carbon nanotubes using any of such substrates by the same method as the method of the present example described above.

(2) Immobilization of DNA by electrostatic binding S having carbon nanotubes produced in (1) above
A DNA probe labeled with a fluorescent labeling substance by electrostatic binding (hereinafter referred to as a fluorescent labeled DNA probe) is immobilized on the i substrate.

First, S containing carbon nanotubes
i The substrate surface is washed with ethanol and distilled water. afterwards,
Treat the surface with a polylysine solution. Separately from this, the fluorescence-labeled DNA probe to be immobilized is washed with ethanol and dispersed in 3-fold citric acid physiological saline.

Then, a citric acid physiological saline solution in which the fluorescent-labeled DNA probe is dispersed is dropped on the surface treated with polylysine. As a result, the phosphate anion contained in the fluorescently labeled DNA probe is electrostatically bound to the cation of polylysine.

Thereafter, the surface is washed with a 0.2% sodium dodecyl sulfate solution, and further washed with 95% ethanol. Next, the immobilized fluorescent labeled DNA probe
Heat at 5 ° C to denature and UV crosslink treatment (60
mJ / cm ² ). Then, as a blocking treatment, 70 mM succinic anhydride and 35% 1-methyl-2-
Pyrrolidinone was dissolved in 0.1M (p
The substrate is immersed in a boric acid solution of H8.0). afterwards,
Let stand for 30 minutes at room temperature. Further, this is thoroughly dried with a dryer at 37 ° C. Then, TE buffer (10 mM Tr
Wash with is-HCl, pH 8.0, containing 0.1 mM EDTA) several times and dry well.

Through the above steps, D as shown in FIG. 9d is obtained.
A probe fixed support including an NA probe is manufactured.

Next, this was washed with water to obtain the unreacted fluorescent label D.
After removing the NA probe, ultraviolet light is irradiated to observe the fluorescence generated from the fluorescent substance. According to this, a plurality of fluorescences are observed on the substrate. Furthermore, by measuring the binding density of the probe, it can be confirmed that about 10 ¹¹ to 10 ¹³ molecules / cm ² of the labeled DNA probe are immobilized on the substrate.

Considering the high aspect ratio of the carbon nanotubes standing almost perpendicular to the substrate, fixing of the DNA probe on the probe fixing support manufactured by the above method is more difficult than fixing on a conventional plane. And 10 ² to 10 ³ times as many D's per equal area
The NA probe can be immobilized. Therefore, by using such a probe-immobilized support of the present invention, it is possible to remarkably improve the sensitivity when detecting a target substance by a measuring method using a fluorescent substance or an electric measuring method.

In the solid phase method of the DNA probe described above, the shape of the long and narrow carrier, that is, linear, fibrous or columnar shape is selected without selecting the material such as the inorganic material, the organic material and the metal. Without being, it can be widely used for producing the probe fixed support of the present invention.

(3) Immobilization of DNA probe by coordinate bond The fluorescent labeled DNA probe is immobilized by coordinate bond on the Si substrate having the carbon nanotubes produced in (1) above.

Electron gun vapor deposition (or film forming techniques such as sputtering, CVD, electrolytic plating and electroless plating may be used) for the carbon nanotubes provided on the Si substrate manufactured in (1) above. Then, an Au film is formed on the surface. On the other hand, separately for the DNA probe, S
(Sulfur) is introduced. For the carbon nanotubes having the film thus formed on the surface thereof, DNA is prepared by the following method.
Coordinate the probe.

First, for the fluorescently labeled DNA probe,
S is introduced as a coordinate bond donor. Next, the fluorescently labeled DNA probe into which S has been introduced is dispersed in a citric acid physiological saline solution. This solution was added to the S containing Au film formed above.
Drop onto the surface of the i substrate. As a result, Au formed on the surface of the carbon nanotube and S contained in the probe are coordinate-bonded. By such a method, the DNA probe is fixed to the elongated carrier by the coordinate bond.

Thereafter, as a blocking treatment, alkanethiol (otherwise, a carbon chain having sulfur, mercaptoethanol or mercaptohexanol, etc. may be used).
The surface of the carbon nanotubes and the surface of the substrate, which are not involved in the coordination bond, are blocked by the treatment.

Next, this was washed with water to obtain unreacted fluorescent label D.
After removing the NA probe and observing the fluorescence generated from the fluorescent substance by irradiating with ultraviolet light, a plurality of fluorescences are observed on the substrate. Furthermore, if the binding density of the probe is measured, a labeled DNA probe of about 10 ¹¹ to 10 ¹³ molecules / cm ² is fixed on the substrate.

Considering the high aspect ratio of the carbon nanotubes standing almost perpendicular to the substrate, fixing the DNA probe on the probe fixing support manufactured by the above method is more difficult than fixing on a conventional plane. And 10 ² to 10 ³ times as many DNAs per equal area
Can be fixed. By using such a probe-immobilized support of the present invention, it is possible to significantly improve the sensitivity when detecting a target substance by a measuring method using a fluorescent substance or an electric measuring method.

Also, in the solid phase method of such a DNA probe, the shape of the long and narrow carrier, that is, the linear, fibrous or columnar shape is selected without selecting the material such as the inorganic material, the organic material and the metal. Can be widely used for producing the probe-immobilized support of the present invention.

In the above example, the labeled probe is used to show that the probe is immobilized at a high density on the probe-immobilized support. However, in the actual use,
Labeling is not necessary.

Example 2. Carbon Nanotubes Grown from SiC Substrate (1) Synthesis of Carbon Nanotubes on SiC Substrate Carbon nanotubes as long and narrow carriers can be produced from a SiC substrate as follows. In the method according to the present embodiment, as shown in FIG. 11a, first, Si (silicon) is formed on the surface.
A SiC substrate 101 having a surface and a back surface having a C (carbon) surface is prepared. Next, the SiC substrate 101 is
CA wash (ie, H ₂ O ₂ + H ₂ SO ₄ wash, water wash, dilute HF wash, water wash, NH ₄ OH + H ₂ O ₂ + H ₂ O wash,
Washing with water, dilute HF washing, water washing, HCl + H ₂ O ₂ + H ₂ O washing, water washing, dilute HF washing in this order).

Next, the SiC substrate 101 is loaded into an electric furnace equipped with an induction heating type carbon heater. Then, the pressure inside the electric furnace was reduced to 10 × 10 ⁻⁴ Torr or less, and
The temperature is raised to 00 to 1800 ° C.

When such vacuum heat treatment is performed, first,
Si atoms are selectively desorbed from the carbon surface of the SiC substrate. Along with this, C which is a bond due to desorption of Si
The atoms recombine with each other (Fig. 11b). As a result of the further reaction, as shown in FIG. 11c, a large number of carbon nanotubes 103 extending from the C surface of the SiC substrate 101 are formed (FIG. 11c). As described above, the carbon nanotubes 103 extending from the surface of the SiC substrate 101
The support 104 is formed.

The carbon nanotubes formed here have a diameter of about 5 to 10 nm, a length of several μm and a density of 10 nm.
^{11-10 13} This is a / cm ^2. The carbon nanotubes are in close contact with each other and the DN is attached to the side of the carbon nanotube.
A biological substance such as A cannot be bound. From this, it is understood that the carbon nanotubes formed in this example exist as an aggregate. FIG. 12 shows a schematic view of the SEM image of the carbon nanotubes thus obtained. It is observed that all carbon nanotubes are growing concentrically (FIG. 12).

DNA was electrostatically bound to the carbon nanotubes in the same manner as in (2) of Example 1 above.
To fix.

(2) Immobilization of DNA by electrostatic binding S having the carbon nanotube produced in (1) above
Fluorescently labeled DNA is immobilized on the iC substrate by electrostatic coupling.

First, S containing carbon nanotubes
The iC substrate surface is washed with ethanol and distilled water. Then, the surface is treated with a polylysine solution. Apart from that,
The fluorescence-labeled DNA to be immobilized is washed with ethanol,
Disperse in 3 times citric acid saline.

Then, a citric acid physiological saline solution in which the fluorescently labeled DNA is dispersed is dropped on the polylysine surface-treated surface. As a result, the phosphate anion contained in the fluorescently labeled DNA is electrostatically bound to the cation of polylysine.

Thereafter, the surface is washed with a 0.2% sodium dodecyl sulfate solution, and further washed with 95% ethanol. Next, the fixed fluorescent-labeled DNA is heated at 95 ° C. to denature it, and UV cross-linking treatment (60 mJ / c
m ² ). Then, as a blocking process, 70
mM succinic anhydride and 35% 1-methyl-2-pyrr
0.1M (pH 8.0) in which olidinone was dissolved
Immerse the substrate in the boric acid solution. Then 30 at room temperature
Leave for a minute. This is thoroughly dried in a dryer at 37 ° C. Then TE buffer (10 mM Tris-HCl
Wash several times (pH 8.0, containing 0.1 mM EDTA) and dry well.

Through the above steps, a probe-immobilized support including a DNA probe as shown in FIG. 11e is manufactured.

Next, this was washed with water and unreacted fluorescent label D
After removing NA, when ultraviolet light is irradiated to observe the fluorescence generated from the fluorescent substance, a plurality of fluorescences are observed on the substrate. Furthermore, if the binding density of the probe is measured, it is 10
It can be seen that about ¹¹ to 10 ¹³ molecules / cm ² of labeled DNA are immobilized on the substrate.

Considering the high aspect ratio of the carbon nanotubes standing almost perpendicular to the substrate, DNA is immobilized on the probe immobilization support prepared by the above method as compared with conventional immobilization on a flat surface. , 10 ² to 10 ³ times as many DNAs can be immobilized per equal area. Therefore, by using such a probe-immobilized support of the present invention, it is possible to remarkably improve the sensitivity when detecting a target substance by a measuring method using a fluorescent substance or an electric measuring method.

In addition, the above-described DNA solid phase method does not require selection of materials such as inorganic substances, organic substances and metals, and
It can be widely used for producing the probe-immobilized support of the present invention without selecting the shape of the long and narrow carrier, that is, the linear shape, the fibrous shape, or the columnar shape.

(3) Immobilization of DNA by coordination bond Fluorescently labeled DNA is immobilized by coordination bond on the SiC substrate having carbon nanotubes produced in (1) above.

Electron gun vapor deposition (or film forming techniques such as sputtering, CVD, electrolytic plating and electroless plating may be used) for the carbon nanotubes provided on the SiC substrate manufactured in (1) above. Then, an Au film is formed on the surface. On the other hand, separately for the DNA probe, S
Have been introduced. A DNA probe is coordinate-bonded to the carbon nanotube having the film thus formed on the surface by the following method.

First, for the fluorescently labeled DNA probe,
S (sulfur) is introduced as a coordinate bond donor. Then S
The fluorescence-labeled DNA probe introduced with is dispersed in a citric acid physiological saline solution. This solution is dropped onto the surface of the SiC substrate having Au formed as described above. As a result, Au formed on the surface of the carbon nanotube and S contained in the probe are coordinate-bonded. By such a method, the DNA probe is fixed to the elongated carrier by the coordinate bond.

Then, as a blocking treatment, alkanethiol (otherwise, a carbon chain having sulfur, mercaptoethanol or mercaptohexanol, etc. may be used).
The surface of the carbon nanotubes and the surface of the substrate which are not involved in the coordination bond are blocked by the treatment.

Next, this was washed with water and unreacted fluorescent label D was added.
After removing the NA probe and observing the fluorescence generated from the fluorescent substance by irradiating with ultraviolet light, a plurality of fluorescences are observed on the substrate. Furthermore, if the binding density of the probe is measured, about 10 ¹¹ to 10 ¹³ molecules / cm ² of labeled DNA probe will be immobilized on the substrate.

Considering the high aspect ratio of the carbon nanotubes standing almost perpendicular to the substrate, fixing the DNA probe on the probe fixing support manufactured by the above method is more difficult than fixing on a conventional plane. And 10 ² to 10 ³ times as many DNAs per equal area
Can be fixed. By using such a probe-immobilized support of the present invention, it is possible to significantly improve the sensitivity when detecting a target substance by a measuring method using a fluorescent substance or an electric measuring method.

Also, in the solid phase method of such a DNA probe, the shape of the elongated carrier, that is, the linear, fibrous or columnar shape is selected without selecting the material such as the inorganic material, the organic material and the metal. Can be widely used for producing the probe-immobilized support of the present invention.

[0211] In the above two examples, the labeled probe was used to show that the probe was immobilized at a high density on the probe-immobilized support. However, in the actual use, labeling was not performed. Not necessarily required.

Example 3. Open-ended carbon nanotube (1) Production of open-ended carbon nanotube The tip of the carbon nanotube provided on the support produced as described in Example 2 above is opened, and the nucleic acid probe is fixed thereto.

First, the carbon nanotubes 120 (FIG. 13a) provided on the SiC substrate 121 manufactured as described in Example 2 above are heated in dry air at 420 ° C. for 30 minutes. As a result, the tip of the carbon nanotube is opened, and the open carbon nanotube 122 is obtained. Moreover, the obtained carbon nanotubes are modified with a carboxyl group.

Next, a DNA probe is fixed to the SiC substrate 123 having the carbon nanotube 122 with the open end by the following method.

(2) Immobilization of DNA probe by electrostatic coupling S having carbon nanotube produced in (1) above
A fluorescence-labeled DNA probe is immobilized on the iC substrate by electrostatic coupling.

First, S containing carbon nanotubes
The iC substrate surface is washed with ethanol and distilled water. Then, the surface is treated with a polylysine solution. Apart from that,
The fluorescence-labeled DNA probe to be immobilized is washed with ethanol and dispersed in 3 times citric acid physiological saline.

Then, a citric acid physiological saline solution in which the fluorescent-labeled DNA probe is dispersed is dropped on the polylysine surface-treated surface. This electrostatically binds the phosphate anion contained in the fluorescently labeled DNA probe to the cation of polylysine.

Then, the surface is washed with a 0.2% sodium dodecyl sulfate solution, and further washed with 95% ethanol. Next, the immobilized fluorescent labeled DNA probe
Heat at 5 ° C to denature and UV crosslink treatment (60
mJ / cm ² ). Then, as a blocking treatment, 70 mM succinic anhydride and 35% 1-methyl-2-
Pyrrolidone dissolved in 0.1M (pH
The substrate is immersed in the boric acid solution of 8.0). Then, it is left at room temperature for 30 minutes. This is thoroughly dried in a dryer at 37 ° C. Then, TE buffer (10 mM Tris-
Wash several times with HCl (pH 8.0) containing 0.1 mM EDTA) and dry well.

Through the above steps, the probe-immobilized support having the DNA probe 124 as shown in FIG. 13c is manufactured.

Next, this was washed with water to obtain the unreacted fluorescent label D.
After removing the NA probe, ultraviolet light is irradiated to observe the fluorescence generated from the fluorescent substance. According to this, a plurality of fluorescences are observed on the substrate. Furthermore, if the binding density of the probe is measured, labeled DNA of about 10 ¹² molecules / cm ² will be obtained.
It can be seen that the probe is fixed on the substrate.

The solid phase method of the DNA probe described above can be performed without selecting materials such as inorganic substances, organic substances and metals.
Further, it can be widely used for producing the probe-immobilized support of the present invention without selecting the shape of the elongated carrier, that is, the linear shape, the fibrous shape or the columnar shape.

(3) Immobilization of DNA probe by coordination bond Fluorescently labeled DNA is immobilized by coordination bond on the SiC substrate having carbon nanotubes produced in (1) above.

Electron gun vapor deposition (or film forming techniques such as sputtering, CVD, electrolytic plating and electroless plating may be used) on the carbon nanotubes provided on the SiC substrate manufactured in (1) above. Then, an Au film is formed on the surface. On the other hand, separately for the DNA probe, S
Have been introduced. A DNA probe is coordinate-bonded to the carbon nanotube having the film thus formed on the surface by the following method.

First, for the fluorescently labeled DNA probe,
S (sulfur) is introduced as a coordinate bond donor. Then S
The fluorescence-labeled DNA probe introduced with is dispersed in a citric acid physiological saline solution. This solution is dropped onto the surface of the SiC substrate having Au formed as described above. As a result, Au formed on the surface of the carbon nanotube and S contained in the probe are coordinate-bonded. By such a method, the DNA probe is fixed to the elongated carrier by the coordinate bond.

After that, as a blocking treatment, alkanethiol (otherwise, a carbon chain having sulfur, mercaptoethanol, mercaptohexanol, or the like may be used).
The surface of the carbon nanotubes and the surface of the substrate, which are not involved in the coordination bond, are blocked by the treatment.

Next, this was washed with water to obtain unreacted fluorescent label D.
After removing the NA probe and observing the fluorescence generated from the fluorescent substance by irradiating with ultraviolet light, a plurality of fluorescences are observed on the substrate. Furthermore, if the binding density of the probe is measured, a labeled DNA probe of about 10 ¹¹ to 10 ¹³ molecules / cm ² is fixed on the substrate.

Considering the high aspect ratio of the carbon nanotubes standing almost perpendicular to the substrate, fixing the DNA probe on the probe fixing support manufactured by the above method is more difficult than fixing on a conventional plane. And 10 ² to 10 ³ times as many DNAs per equal area
Can be fixed. By using such a probe-immobilized support of the present invention, it is possible to significantly improve the sensitivity when detecting a target substance by a measuring method using a fluorescent substance or an electric measuring method.

Also, in the solid phase method of such a DNA probe, the shape of the elongated carrier, that is, the linear shape, the fibrous shape or the columnar shape is selected without selecting the material such as the inorganic material, the organic material and the metal. Can be widely used for producing the probe-immobilized support of the present invention.

In the above two examples, labeled probes were used to show that the probes are immobilized at high density on the probe-immobilized support. However, in the actual use, labeling is not performed. Not necessarily required.

(4) Immobilization of DNA probe by covalent bond Since a functional group is introduced into the opening of the opened carbon nanotube, it is possible to solidify the probe by utilizing the functional group. . An example is given below.

First, carbon nanotubes are grown on a substrate by the method described in (1). Next, the carbon nanotubes are heated in a dry air atmosphere. As a result, the ends of the carbon nanotubes are opened, and open-ended carbon nanotubes are obtained. A -COOH group is introduced at the edge of the opening. After passing this —COOH group via —SOCl ₂ and —COCl ₂ ,
"(Hight carrier) -CONH- (Probe)" immobilized by covalently form of is achieved by substitution reactions with probes introduced and H ₂ group. This reaction can be carried out by utilizing a chemical reaction known per se.

At this time, by adding a fluorescent substance to the DNA probe to be immobilized, it is possible to confirm the amount and density of the immobilization.

For example, the carbon nanotubes on which the labeled probe is immobilized are washed with water to remove the unreacted DNA probe and then irradiated with ultraviolet light to detect the fluorescence from the fluorescent substance and measure the binding density of the DNA probe. . According to it, DNA of about 10 ¹¹ to 10 ¹³ molecules / cm ²
It can be seen that the probe is fixed.

Here, an example of using a labeled probe was shown to show that the probe is immobilized at a high density on the probe-immobilized support, but in the actual use, labeling is not always necessary. .

Example 4. Using carbon nanotubes with a carbon nanotube diameter of 6 mm, which is not fixed to the substrate, 500 Torr
Carbon nanotubes present in carbon deposited on the cathode (about 5 nm in diameter, about 5 μm in length) after applying a voltage of 10 V in the He gas atmosphere to cause arc discharge
Is obtained. It is possible to immobilize the nucleic acid probe on the thus obtained carbon nanotube by the method according to any one of the above examples. The probe-immobilized carrier thus produced is also preferably used.

Immobilization of the DNA probe on the carbon nanotubes produced as described above can be carried out by using any of the methods described in Examples 1 and 2 above. Further, the tip can be opened by the method described in Example 3, and furthermore, the probe can be immobilized on the opened carbon nanotube by using the method described in Example 3.

Example 5. ZnO columnar crystal (1) Synthesis of ZnO columnar crystal An α-Al ₂ O ₃ substrate having a crystal face of (0001) [hereinafter referred to as (0001) α-Al ₂ O ₃ substrate] was treated with trichloroethane, acetone, and ethanol. After ultrasonic cleaning using, it is cleaned with ion-exchanged water for 30 minutes. This board is CV
Introduced into the film forming chamber of D device, and heated to 550 ° C
Heat to. Next, 99.9% of zinc-2,4-pe
The tannedionate is heated to 115 ° C. to evaporate, and the same amount of oxygen gas as nitrogen gas of 1.2 dm ³ / min is introduced into the film forming chamber as a carrier. The distance between the inlet to the film forming chamber and the (0001) α-Al ₂ O ₃ substrate was 5 m.
m, and is sprayed directly on the (0001) α-Al ₂ O ₃ substrate, and ZnO is formed into a film by thermal decomposition.

At a film forming speed of 200 nm / min, about 500
When a film is formed for 10 minutes, a ZnO columnar crystal having a length of 100 μm and a diameter of 1.5 μm, which is aligned in the direction perpendicular to the substrate, is obtained. FIG. 15 shows a schematic view of ZnO columnar crystals when observed by SEM. The number density of these columnar crystals is 500,000 / mm
It is ² .

(2) Immobilization of DNA probe by electrostatic coupling The ZnO columnar crystal prepared in the above (1) is provided (00
01) Fluorescently labeled DNA probe is immobilized on the α-Al ₂ O ₃ substrate by electrostatic coupling.

First, it has ZnO columnar crystals (000
1) The α-Al ₂ O ₃ substrate is washed with ethanol and distilled water. Then, the surface is treated with a polylysine solution. Separately from this, the fluorescence-labeled DNA probe to be immobilized is washed with ethanol and dispersed in 3-fold citric acid physiological saline.

Then, a citric acid physiological saline solution in which the fluorescent-labeled DNA probe is dispersed is dropped on the surface treated with polylysine. As a result, the phosphate anion contained in the fluorescently labeled DNA probe is electrostatically bound to the cation of polylysine.

Then, the surface is washed with a 0.2% sodium dodecylsulfate solution, and further washed with 95% ethanol. Next, the fixed fluorescent-labeled DNA is heated at 95 ° C. to denature it, and UV cross-linking treatment (60 mJ / c
m ² ). Then, as a blocking process, 70
mM succinic anhydride and 35% 1-methyl-2-pyrr
0.1M (pH8.
The substrate is immersed in the boric acid solution of 0). Then, it is left at room temperature for 30 minutes. This is thoroughly dried in a dryer at 37 ° C. Then TE buffer (10 mM Tris-HC
1 (pH 8.0) containing 0.1 mM EDTA)
Wash several times with and dry well.

Through the above steps, a probe-immobilized support having a DNA probe is manufactured. A fluorescent labeled DNA probe is attached. The columnar ZnO was washed with water to remove unreacted DNA probe, and then ultraviolet light was irradiated to cause the fluorescent material to emit light.
It will be understood that about ¹¹ to 10 ¹³ molecules / cm ² of DNA probe are immobilized.

Due to the high aspect ratio of ZnO standing almost perpendicular to the substrate, this probe-immobilized support immobilizes 10 ² to 10 ³ times as many DNA probes per equivalent area as compared to conventional binding to a flat surface. can do. This improves the sensitivity when performing the measuring method using the fluorescent substance or the electric measuring method.

Although the ZnO columnar crystal is described in this example, the probe can be solid-phased on the diamond columnar crystal by a similar method. That is, if the growth direction is substantially perpendicular to the substrate, it is possible to immobilize the DNA probe, which is an example of the nucleic acid probe, on the long and thin simple substance made of different materials by the same method.

(3) Immobilization of DNA probe by coordination bond The ZnO columnar crystal prepared in (1) above (00)
01) Fluorescently labeled DNA is immobilized on the α-Al ₂ O ₃ substrate by a coordinate bond.

The (0001) α-produced in the above (1)
For the ZnO columnar crystals provided on the Al ₂ O ₃ substrate,
Electron gun vapor deposition (or a film forming technique such as sputtering, CVD, electrolytic plating and electroless plating may be used) is performed to form an Au film on the surface. On the other hand, S is separately introduced into the DNA probe. A DNA probe is coordinate-bonded to the ZnO columnar crystal having the film thus formed on the surface by the following method.

First, for the fluorescently labeled DNA probe,
S is introduced as a coordinate bond donor. Next, the fluorescently labeled DNA probe into which S has been introduced is dispersed in a citric acid physiological saline solution. This solution is dripped onto the surface of the (0001) α-Al ₂ O ₃ substrate having Au formed as described above. As a result, A formed on the surface of the columnar crystal of ZnO
u and S provided on the probe are coordinate-bonded. By such a method, the DNA probe is fixed to the elongated carrier by the coordinate bond.

Then, as a blocking treatment, alkanethiol (otherwise, a carbon chain having sulfur, mercaptoethanol or mercaptohexanol, etc. may be used).
To block the ZnO columnar crystal surface and the substrate surface that did not participate in the coordination bond.

Next, this was washed with water to obtain the unreacted fluorescent label D.
After removing the NA probe and observing the fluorescence generated from the fluorescent substance by irradiating with ultraviolet light, a plurality of fluorescences are observed on the substrate. Furthermore, if the binding density of the probe is measured, labeled DNA of about 10 ¹¹ to 10 ¹³ molecules / cm ²
The probe is fixed on the substrate.

[0251] Further, the substrate stands almost vertically.
Considering the high aspect ratio of ZnO columnar crystals
On a probe fixed support manufactured by the method
Fixing of the A probe is more difficult than fixing on a conventional plane.
10 per equal area^Two-10 ^ThreeFix double the number of DNA
can do. Therefore, such a plot of the present invention
If a fixed support is used, a measurement method using a fluorescent substance
Sense when detecting a target substance by an electric or electrical measurement method
It is possible to significantly improve the degree.

In the above two examples, the labeled probe was used to show that the probe was immobilized at a high density on the probe-immobilized support. However, in the actual use, labeling was not performed. Not necessarily required.

Although the ZnO columnar crystal is described in this example, the probe can be solid-phased on the diamond columnar crystal by a similar method. That is, as long as the growth direction is substantially perpendicular to the substrate, it is possible to immobilize the nucleic acid probe on the long and thin simple substance made of different materials by the same method.

Example 6. Fibrous material of Si ₃ fibrous material (1) of N _{4 Si} 3 production of fibrous material _{N 4 Si} 3 _{N 4} is manufactured as follows. A quartz substrate is coated with a chloride solution of Ni, Fe, or Co and dried in a hydrogen atmosphere while heating the substrate. As a result, fine metal particles having a diameter on the order of submicrons are dispersed on the surface of the substrate. This substrate is introduced into a CVD device and heated to 1200 ° C. Next, silane gas and hydrogen gas with hydrogen gas as a carrier are introduced into this CVD apparatus and reacted for 30 minutes. With such a method, Si ₃
N ₄ fibrous material is formed. The fibrous substance produced in this manner has a length of about 1 mm and a diameter of 0.5.
~ 2 μm.

(2) Immobilization of DNA Probe by Electrostatic Bonding Fluorescently labeled DNA probe is immobilized by electrostatic bonding on the quartz substrate having the Si ₃ N ₄ fibrous substance produced in (1) above.

First, the quartz substrate having the Si ₃ N ₄ fibrous substance is washed with ethanol and distilled water. Then, the surface is treated with a polylysine solution. Separately from this, the fluorescence-labeled DNA probe to be immobilized is washed with ethanol and dispersed in 3-fold citric acid physiological saline.

After that, a citric acid physiological saline solution in which the fluorescent-labeled DNA probe is dispersed is dropped on the surface treated with polylysine. As a result, the phosphate anion contained in the fluorescently labeled DNA probe is electrostatically bound to the cation of polylysine.

Then, the surface is washed with a 0.2% sodium dodecyl sulfate solution, and further washed with 95% ethanol. Next, the immobilized fluorescent labeled DNA probe
Heat at 5 ° C to denature and UV crosslink treatment (60
mJ / cm ² ). Then, as a blocking treatment, 70 mM succinic anhydride and 35% 1-methyl-2-
Pyrrolidone dissolved in 0.1M (pH
The substrate is immersed in the boric acid solution of 8.0). Then, it is left at room temperature for 30 minutes. This is thoroughly dried in a dryer at 37 ° C. Then TE buffer (10 mM Tris
Wash several times with HCl (pH 8.0) containing 0.1 mM EDTA) and dry well.

Through the above steps, a probe-immobilized support having a DNA probe is manufactured. This fibrous Si
₃ N ₄ was washed with water to remove the unreacted DNA probe, and then ultraviolet light was irradiated to cause the fluorescent material to emit light. When the binding density was measured, about 10 ¹¹ to 10 ¹³ molecules / cm ² of DNA probe were fixed. I know that

(3) Immobilization of DNA probe by coordination bond A DNA probe labeled with a fluorescent substance by coordination bond is attached to the quartz substrate having the Si ₃ N ₄ fibrous material produced in (1) above. Fix it.

Provided on the quartz substrate manufactured in (1) above.
Si_ThreeN_FourElectron gun evaporation (or
Is sputtering, CVD, electrolytic plating, electroless plating, etc.
Film forming technique may be used) to form an Au film on the surface.
To film. On the other hand, separately for the DNA probe, S
Have been introduced. Si having a film formed on the surface in this way _Three
N_FourDNA of the fibrous substance of
Coordinate the probe.

First, for the fluorescently labeled DNA probe,
S (sulfur) is introduced as a coordinate bond donor. Then S
The fluorescence-labeled DNA probe introduced with is dispersed in a citric acid physiological saline solution. This solution is dropped on the surface of the quartz substrate having Au formed as described above. This makes S
Au formed as a film on the surface of the fibrous substance of i ₃ N ₄ and S contained in the probe are coordinate-bonded. By such a method, the DNA probe is fixed to the elongated carrier by the coordinate bond.

Then, as a blocking treatment, alkanethiol (otherwise, a carbon chain having sulfur, mercaptoethanol or mercaptohexanol, etc. may be used).
The surface of the Si ₃ N ₄ fibrous material not involved in the coordination bond and the surface of the substrate are blocked.

Next, this was washed with water and unreacted fluorescent label D was added.
After removing the NA probe and observing the fluorescence generated from the fluorescent substance by irradiating with ultraviolet light, a plurality of fluorescences are observed on the substrate. Furthermore, if the binding density of the probe is measured, about 10 ¹¹ to 10 ¹³ molecules / cm ² of labeled DNA probe will be immobilized on the substrate.

[0265] Further, the substrate stands almost vertically.
Si_ThreeN_FourThe high aspect ratio of various fibrous materials
And the probe fixing support manufactured by the above method.
The DNA probe is fixed on a conventional flat surface.
Compared to 10 per equivalent area ^Two-10^ThreeDouble the number of DNA
Can be fixed. Such a probe of the present invention
If you use a fixed support, you can
Sensitivity when detecting a target substance by an electrical measurement method
Can be significantly improved.

In the above two examples, the labeled probe was used to show that the probe was immobilized at a high density on the probe-immobilized support. However, in the actual use, labeling was not performed. Not necessarily required.

In addition, Si ₃ N synthesized without being oriented to the substrate
Similarly, for ₄ fibrous crystals, a DNA probe can be bound by using binding methods such as electrostatic binding and coordinate binding.

Further, a DNA probe is fixed to fibrous substances such as magnesium sulfate hydroxide whiskers and gypsum whiskers obtained by dehydration of gypsum dihydrate by the same method as in the case of Si ₃ N ₄ fibrous crystals. It is possible.

Example 7. Long thin support the Si ₃ N ₄ spiral material (1) _Si 3 _{N 4} spiral substances produced _Si 3 _{N 4} of the spiral-like substance can be prepared as follows. That is, a chloride solution of Ni or Fe is applied to a graphite substrate and dried in a hydrogen atmosphere while heating the substrate to disperse metal fine particles having a diameter of the submicron order on the substrate surface. This substrate is introduced into a CVD device and heated to a temperature of 1200 ° C. Next, this CV
Silane gas using hydrogen gas as a carrier and ammonia gas are introduced into the apparatus D and reacted for 30 minutes. By this method, a spiral material of Si ₃ N ₄ is formed. The diameter of each spiral material is 0.5 to 2 μm, and the spiral pitch is 3 to 5 μm.
m, and the spiral diameter is 10 to 15 μm.

(2) Immobilization of DNA probe by electrostatic binding [0270] Fluorescent labeling D was performed by electrostatic binding on the graphite substrate having the Si ₃ N ₄ helical substance produced in (1) above.
Fix the NA probe.

First, a graphite substrate having a Si ₃ N ₄ spiral material is washed with ethanol and distilled water. Then, the surface is treated with a polylysine solution. Separately from this, the fluorescence-labeled DNA probe to be immobilized is washed with ethanol and dispersed in 3-fold citric acid physiological saline.

Then, a citric acid physiological saline solution in which the fluorescent-labeled DNA probe is dispersed is dropped on the surface treated with polylysine. As a result, the phosphate anion contained in the fluorescently labeled DNA probe is electrostatically bound to the cation of polylysine.

After that, the surface is washed with a 0.2% sodium dodecyl sulfate solution, and further washed with 95% ethanol. Next, the immobilized fluorescent labeled DNA probe
Heat at 5 ° C to denature and UV crosslink treatment (60
mJ / cm ² ). Then, as a blocking treatment, 70 mM succinic anhydride and 35% 1-methyl-2-
The substrate is immersed in a 0.1 M (pH 8.0) boric acid solution in which pyrrolidinone is dissolved. Then, it is left at room temperature for 30 minutes. This is thoroughly dried in a dryer at 37 ° C. Then TE buffer (10 mM Tris-HCl, pH
8.0, containing 0.1 mM EDTA) and dried several times.

Through the above steps, the probe-immobilized support having the DNA probe is manufactured.

The spiral Si ₃ N ₄ is washed with water to remove unreacted DNA probe, and then ultraviolet light is irradiated to cause the fluorescent material to emit light, and the binding density is measured. According to it, 10 ¹¹
It can be seen that DNA probes of about 10 ¹³ molecules / cm ² are immobilized.

(3) Immobilization of DNA Probe by Coordination Bonding The graphite substrate having the Si ₃ N ₄ helical substance produced in (1) above was coordinate-bonded to give a fluorescent-labeled DNA probe. Fix it.

Electron gun deposition (or film forming techniques such as sputtering, CVD, electrolytic plating and electroless plating) on the Si ₃ N ₄ spiral material provided on the graphite substrate manufactured in (1) above. May be used) and A
A u film is formed. On the other hand, S is separately introduced into the DNA probe. A DNA probe is coordinate-bonded to the helical substance of Si ₃ N ₄ having a film formed on the surface thereof by the following method.

First, for the fluorescently labeled DNA probe,
S (sulfur) is introduced as a coordinate bond donor. Then S
The fluorescence-labeled DNA probe introduced with is dispersed in a citric acid physiological saline solution. This solution is dropped on the surface of the graphite substrate having Au formed as described above. As a result, Au formed as a film on the surface of the Si ₃ N ₄ spiral material and S contained in the probe are coordinate-bonded. By such a method, the DNA probe is fixed to the elongated carrier by the coordinate bond.

After that, as a blocking treatment, alkanethiol (otherwise, a carbon chain having sulfur, mercaptoethanol, mercaptohexanol, or the like may be used).
To block the surface of the Si ₃ N ₄ spiral material that does not participate in the coordination bond and the surface of the substrate.

Next, this was washed with water to obtain the unreacted fluorescent label D.
After removing the NA probe and observing the fluorescence generated from the fluorescent substance by irradiating with ultraviolet light, a plurality of fluorescences are observed on the substrate. Furthermore, if the binding density of the probe is measured, about 10 ¹¹ to 10 ¹³ molecules / cm ² of labeled DNA probe will be immobilized on the substrate.

In consideration of the high aspect ratio of the Si ₃ N ₄ spiral material whose axis stands substantially perpendicular to the substrate, the DNA probe of the probe-immobilized support produced by the above method is considered. The immobilization can immobilize 10 ² to 10 ³ times as many DNAs per equal area as compared with the conventional immobilization on a flat surface. By using such a probe-immobilized support of the present invention, it is possible to significantly improve the sensitivity when detecting a target substance by a measuring method using a fluorescent substance or an electric measuring method.

In the above two examples, the labeled probe was used to show that the probe is immobilized at a high density on the probe-immobilized support. However, in the actual use, labeling is not performed. Not necessarily required.

Similarly, S synthesized without being oriented to the substrate was used.
It is possible to bind the labeled DNA sample that emits fluorescence to the i ₃ N ₄ helical substance also by using a binding method such as electrostatic binding and coordinate binding.

The method performed for the above Si ₃ N ₄ spiral material can be similarly performed for carbon fibers having a spiral structure. Thereby, the DNA probe can be satisfactorily immobilized.

Example 8. Hydroxyapatite linear substance (1) Manufacturing method: 1 g of β-tricalcium phosphate (β-Ca ₃ (PO ₄ ) ₂ ) and 5 g of pressure under a pressure of 2 MPa and a temperature of 473K.
A linear substance of hydroxyapatite is formed by hydrothermally reacting the aqueous solution containing citric acid (that is, utilizing the property of high temperature and high pressure water). This substance has a structure in which linear crystals radially extend from the center toward the outside like a sea urchin.

Also for this hydroxyapatite, binding methods such as electrostatic bonding, coordination bonding, covalent bonding and adsorption can be used. Thereby it is possible to bind a DNA probe.

(2) Immobilization of DNA Probe by Electrostatic Bonding The fluorescently labeled DNA probe is immobilized by electrostatic bonding on the hydroxyapatite linear substance produced in (1) above.

First, the linear substance of hydroxyapatite is washed with ethanol and distilled water. Then, the surface is treated with a polylysine solution. Separately, the fluorescence-labeled DNA probe to be immobilized was washed with ethanol and washed with 3
Disperse in double citric acid saline.

Then, a citric acid physiological saline solution in which the fluorescence-labeled DNA probe is dispersed is dropped on the polylysine surface-treated surface. As a result, the phosphate anion contained in the fluorescently labeled DNA probe is electrostatically bound to the cation of polylysine.

After that, the surface is washed with a 0.2% sodium dodecyl sulfate solution and further washed with 95% ethanol. Next, the immobilized fluorescent labeled DNA probe
Heat at 5 ° C to denature and UV crosslink treatment (60
mJ / cm ² ). Then, as a blocking treatment, 70 mM succinic anhydride and 35% 1-methyl-2-
Pyrrolidone dissolved in 0.1M (pH
Immerse the hydroxyapatite linear substance in the boric acid solution of 8.0). Then, it is left at room temperature for 30 minutes. This is thoroughly dried in a dryer at 37 ° C. Then, TE buffer (10 mM Tris-HCl (pH
8.0), containing 0.1 mM EDTA) several times and dried well.

By the above steps, the DNA probe was prepared.
A probe fixing support is manufactured. This hydro
After washing the linear substance of xiapatite with water, unreacted DNA
After removing the lobes, the fluorescent material is emitted by irradiating it with ultraviolet light.
And the bond density is measured to be 10 ¹¹-10^ThirteenMolecule / c
m^TwoIt is clear that some DNA probes are fixed
It

(3) Immobilization of DNA probe by coordination bond The linear substance of hydroxyapatite produced in (1) above was labeled with a fluorescent substance by coordination bond.
Fix the NA probe.

Electron gun vapor deposition (or film forming techniques such as sputtering, CVD, electrolytic plating and electroless plating may be used) is applied to the linear substance of hydroxyapatite produced in the above (1). Then, an Au film is formed on the surface.
On the other hand, S is separately introduced into the DNA probe. For the hydroxyapatite linear substance having a film formed on the surface in this manner, D
Coordinate the NA probe.

First, for the fluorescently labeled DNA probe,
S (sulfur) is introduced as a coordinate bond donor. Then S
The fluorescence-labeled DNA probe introduced with is dispersed in a citric acid physiological saline solution. This solution is dropped on the surface of the quartz substrate having Au formed as described above. As a result, A formed on the surface of the linear material of hydroxyapatite
u and S provided on the probe are coordinate-bonded. By such a method, the DNA probe is fixed to the elongated carrier by the coordinate bond.

After that, as a blocking treatment, alkanethiol (otherwise, a carbon chain having sulfur, mercaptoethanol, mercaptohexanol, or the like may be used).
To block the surface of the hydroxyapatite linear substance that does not participate in the coordination bond.

Next, this was washed with water to obtain unreacted fluorescent label D.
After removing the NA probe and observing the fluorescence generated from the fluorescent substance by irradiating it with ultraviolet light, a plurality of fluorescences are observed on the linear substance. Furthermore, if the binding density of the probe is measured, labeled DN of about 10 ¹¹ to 10 ¹³ molecules / cm ²
The A probe would be immobilized on the hydroxyapatite linear material.

(4) Immobilization of DNA probe by covalent bond —OH group is present in the linear substance of hydroxyapatite. Therefore, it is possible to immobilize the probe using this functional group. An example thereof will be described below with reference to FIG. In FIG. 14, a diagram in which one —OH group is present on the surface 131 of the hydroxyapatite linear substance 130 is illustrated for convenience, but the number of —OH groups is not limited to this.

Next, 1% of N-AEPTMS (N-aminoethyl-aminopropyl-trimethoxysilane) is stirred at room temperature for 1 hour. The above hydroxyapatite linear substance is immersed in this solution to form a compound having an amino group on the surface thereof (FIG. 14b). Next, this hydroxyapatite linear substance is washed with pure water, dried with nitrogen gas, and then baked at 120 ° C. Then, it was immersed for 2 hours at room temperature in a solution of 0.3 mg / mL of EMCS (divalent cross-linking agent that reacts with amino group and thiol group) in dimethyl sulfoxide: ethanol = 1: 1. The crosslinker is attached to the amino group. The hydroxyapatite linear substance is washed with ethanol and dried with nitrogen gas (FIG. 14c).

Separately, D labeled with a fluorescent substance
Prepare an NA probe (Fig. 14d). The star in the figure is a fluorescent substance. Td against this fluorescently labeled DNA probe
T (Terminal deosynucleotidyl Transferase) enzyme is allowed to act, and then 4-thio UTP (uridine triphosphate) is allowed to act. This makes it possible to introduce a thiol group into the 3'end of the DNA probe (Fig. 14e).

This hydroxyapatite linear substance is washed with water to remove unreacted DNA probe, and then ultraviolet light is irradiated to detect fluorescence from the fluorescent substance to measure the binding density of the DNA probe. According to it, 10 ¹¹
It can be seen that DNA probes of about 10 ¹³ molecules / cm ² are immobilized.

Considering the high aspect ratio of the radially extending linear substance included in the hydroxyapatite linear substance, immobilization of the DNA probe on the probe-immobilized support produced by the above method is performed by conventional methods. It is possible to immobilize 10 ² to 10 ³ times as many DNAs per equal area as compared to immobilization on a flat surface. By using such a probe-immobilized support of the present invention, it is possible to significantly improve the sensitivity when detecting a target substance by a measuring method using a fluorescent substance or an electric measuring method.

(5) Immobilization of DNA probe by covalent bond 2 Hydroxyapatite whiskers were
It is also possible to immobilize the DNA probe using the OH group as follows. First, 1% of epoxysilane (3-glycidoxypropyltrimethoxysilane) is reacted with the surface of hydroxyapatite whiskers. As a result, H of the —OH group and (CH ₃ O) — of the epoxysilane are eliminated and condensed. As a result, the epoxysilane-derived substance is bonded to the surface of the hydroxyapatite, resulting in epoxysilica.

Next, after making the tip of the epoxy silica a diol, tresyl chloride is reacted to form a tresyl silica.

Separately from this, DNA having an amino group introduced
Prepare the probe. DNA in which amino groups are introduced on the surface of hydroxyapatite modified with tresyl silica
By binding the probe, the DNA probe can be covalently bound to the substrate having the —OH group.

This linear hydroxyapatite is washed with water to remove unreacted DNA probe. After that, ultraviolet light is irradiated to measure the fluorescence from the fluorescent substance, and the binding density of the DNA probe is measured. According to it, 10 ¹¹ to 10
It can be seen that a DNA probe of about ¹³ molecules / cm ² is immobilized on the linear hydroxyapatite.

Due to the linear structure of hydroxyapatite formed radially, it is possible to immobilize 10 ² to 10 ³ times as many DNAs per equal area as compared with the conventional binding to a plane. Therefore, the sensitivity when performing a measuring method using a fluorescent substance or an electrical measuring method is improved.

In this example, the -OH group existing in hydroxyapatite was used to carry out the covalent bond. However, a method in which a DNA probe can be covalently bonded to the hydroxyapatite surface by a conventional method known per se is also used. It is possible to

In the above four examples, the labeled probe was used to show that the probe was immobilized at a high density on the probe-immobilized support. However, in the actual use, labeling was not performed. Not necessarily required.

Example 9. Metal Linear Crystal (1) Manufacturing Method A gold (Au) linear crystal as an example of a metal linear crystal can be synthesized by the following reduction method.

A boat filled with gold halide is introduced into a quartz tube covered with an electric furnace and then heated to 550 ° C. After the reduction reaction by heat is completed, the quartz tube is cooled and helium gas is flown into the quartz tube. By this method, gold linear crystals can be obtained.

Also for this gold linear crystal, a coupling method such as electrostatic coupling or coordination coupling is used in the same manner as described above.
It is possible to attach a labeled DNA probe that fluoresces. The gold linear crystals were washed with water to remove unreacted DNA.
After removing the probe, ultraviolet light is irradiated and the fluorescence from the fluorescent substance is measured. According to this, it is found that about 10 ¹¹ to 10 ¹³ molecules / cm ² of DNA probe are immobilized on the gold linear crystal.

(2) Immobilization of DNA probe by electrostatic bond A fluorescent labeled DNA probe is immobilized by electrostatic bond to the gold linear crystal produced in (1) above.

First, gold linear crystals are washed with ethanol and distilled water. Then, the surface is treated with a polylysine solution. Separately from this, the fluorescence-labeled DNA probe to be immobilized is washed with ethanol and dispersed in 3-fold citric acid physiological saline.

Then, a citric acid physiological saline solution in which the fluorescent-labeled DNA probe is dispersed is dropped on the surface treated with polylysine. As a result, the phosphate anion contained in the fluorescently labeled DNA probe is electrostatically bound to the cation of polylysine.

Thereafter, the surface is washed with a 0.2% sodium dodecyl sulfate solution, and further washed with 95% ethanol. Next, the immobilized fluorescent labeled DNA probe
Heat at 5 ° C to denature and UV crosslink treatment (60
mJ / cm ² ). Then, as a blocking treatment, 70 mM succinic anhydride and 35% 1-methyl-2-
Pyrrolidone dissolved in 0.1M (pH
The gold linear crystals are immersed in the boric acid solution of 8.0). Then, it is left at room temperature for 30 minutes. This is thoroughly dried in a dryer at 37 ° C. Then, TE buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDT was used.
(Containing A) and washed well several times.

Through the above steps, the probe-immobilized support having the DNA probe is manufactured. This gold linear crystal is washed with water to remove unreacted DNA probe, and then ultraviolet light is irradiated to cause the fluorescent material to emit light. When the binding density is measured, a DNA probe of about 10 ¹¹ to 10 ¹³ molecules / cm ² is obtained. You can see that it is fixed.

(3) Immobilization of DNA probe by coordination bond A DNA probe labeled with a fluorescent substance by coordination bond is immobilized on the gold linear crystal produced in (1) above.

First, for the fluorescently labeled DNA probe,
S (sulfur) is introduced as a coordinate bond donor. Then S
The fluorescence-labeled DNA probe introduced with is dispersed in a citric acid physiological saline solution. This solution is dropped on the surface of the gold linear crystal formed as described above. As a result, Au on the surface of the gold linear crystal and S contained in the probe are coordinate-bonded. By such a method, the DNA probe is fixed to the elongated carrier by the coordinate bond.

Then, as a blocking treatment, alkanethiol (otherwise, a carbon chain having sulfur, mercaptoethanol, mercaptohexanol, or the like may be used).
To block the surface of the gold linear crystal that does not participate in the coordinate bond.

Next, this was washed with water to obtain the unreacted fluorescent label D.
After removing the NA probe and observing the fluorescence generated from the fluorescent substance by irradiating with ultraviolet light, a plurality of fluorescences are observed on the substrate. Furthermore, if the binding density of the probe is measured, about 10 ¹¹ to 10 ¹³ molecules / cm ² of labeled DNA probe will be immobilized on the gold linear crystal.

In consideration of the high aspect ratio of the gold linear crystal, the fixing of the DNA probe on the probe fixing support manufactured by the above method is performed by 10 ² per equal area as compared with the conventional fixing on a flat surface. It is possible to immobilize 10 to 10 ³ times as many DNAs. By using such a probe-immobilized support of the present invention, it is possible to significantly improve the sensitivity when detecting a target substance by a measuring method using a fluorescent substance or an electric measuring method.

In the above two examples, labeled probes were used to show that the probes are immobilized on the probe-immobilized support at a high density. However, in the actual use, labeling was not performed. Not necessarily required.

Metals which can be used to obtain linear crystals by the above-mentioned reduction method include Cu, Ag, Fe, Ni, Co and Pt in addition to Au. Moreover, if the aggregation method from the gas phase is used,
Ni, Cu, Fe, Pt, Ti, Au, Al, Ba, G
Metal linear crystals of e, V and Ag are obtained. Furthermore, if a thermal decomposition method is used, metal linear crystals of Pd, Pt, Au, etc. can be obtained. Similarly, 10 ² to 10 ³ times as many DNA probes can be immobilized per equivalent area as compared to the case where the immobilization is performed on a conventional plane for these linear crystal form metals. Therefore, the probe-immobilized support and probe-immobilized carrier comprising those metal linear crystals are
Similarly, it is possible to improve the detection sensitivity when performing a measurement method using a fluorescent substance or an electrical measurement method.

In the case of a metal other than Au, in the case where the probe is solid-phased by coordination bond, an electron gun vapor deposition (or sputtering, CVD) is applied to such a long carrier made of metal. It is also possible to perform a film forming technique such as electrolytic plating and electroless plating) to form an Au film on the surface, and similarly, a DNA probe introduced with S is coordinate-bonded.

Example 10. Polyoxymethylene having a needle crystal structure (1) Production method A method for producing needle crystals of an organic substance will be described below. First, the three-necked flask was evacuated, then purged with argon gas and kept at 1 atm. Next, 0.08 mol of trioxane is dissolved in 250 mL of cyclohexane, refluxed with sodium hydride and distilled to 200 mL. Here 5
0 ℃, 10mL trifluoride boron gas and 10mL
Distilled water in 1 above is added, the mixture is stirred vigorously, and the mixture is allowed to stand. Thereby, a needle-like crystal structure of polyoxymethylene can be obtained.

For this polyoxymethylene needle crystal,
The fluorescent substance DNA probe is bound by a binding method such as electrostatic binding or coordinate binding according to the following method.

(2) Immobilization of DNA probe by electrostatic bond A fluorescent labeled DNA probe is immobilized on the polyoxymethylene needle crystals produced in (1) above by electrostatic bond.

First, polyoxymethylene needle crystals are washed with ethanol and distilled water. Then, the surface is treated with a polylysine solution. Separately from this, the fluorescence-labeled DNA probe to be immobilized is washed with ethanol and dispersed in 3-fold citric acid physiological saline.

Then, a citric acid physiological saline solution in which the fluorescent-labeled DNA probe is dispersed is dropped on the surface treated with polylysine. As a result, the phosphate anion contained in the fluorescently labeled DNA probe is electrostatically bound to the cation of polylysine.

Then, the surface is washed with a 0.2% sodium dodecyl sulfate solution, and further washed with 95% ethanol. Next, the immobilized fluorescent labeled DNA probe
Heat at 5 ° C to denature and UV crosslink treatment (60
mJ / cm ² ). Then, as a blocking treatment, 70 mM succinic anhydride and 35% 1-methyl-2-
Pyrrolidone dissolved in 0.1M (pH
The polyoxymethylene needle crystals are immersed in the boric acid solution of 8.0). Then, it is left at room temperature for 30 minutes. This is thoroughly dried in a dryer at 37 ° C. Then, TE buffer (10 mM Tris-HCl (pH 8.0), 0.
Wash with several times (containing 1 mM EDTA) and dry well.

Through the above steps, a probe-immobilized support having a DNA probe is manufactured. This polyoxymethylene needle crystal is washed with water to remove unreacted DNA probe, and then ultraviolet light is irradiated to cause the fluorescent material to emit light, and the binding density is measured to be about 10 ¹¹ to 10 ¹³ molecules / cm ² DNA probe. You can see that is fixed.

(3) Immobilization of DNA probe by coordination bond A DNA probe labeled with a fluorescent substance by coordination bond is immobilized on the polyoxymethylene needle crystal produced in (1) above.

Electron gun deposition (or sputtering, C) was applied to the polyoxymethylene needle crystals produced in (1) above.
A film forming technique such as VD, electrolytic plating or electroless plating may be used) to form an Au film on the surface. On the other hand, S is separately introduced into the DNA probe.
A DNA probe is coordinate-bonded to the needle-like polyoxymethylene crystal having a film formed on the surface thereof by the following method.

Next, for the fluorescently labeled DNA probe,
S (sulfur) is introduced as a coordinate bond donor. Then S
The fluorescence-labeled DNA probe introduced with is dispersed in a citric acid physiological saline solution. This solution is added dropwise to the surface of the polyoxymethylene needle-shaped crystal formed as described above. As a result, Au on the surface of the polyoxymethylene needle crystal and S contained in the probe are coordinate-bonded. By such a method, the DNA probe is fixed to the elongated carrier by the coordinate bond.

Then, as a blocking treatment, alkanethiol (otherwise, a carbon chain having sulfur, mercaptoethanol or mercaptohexanol, etc. may be used).
The polyoxymethylene needle-like crystal surface that is not involved in the coordination bond is blocked with

Next, this was washed with water to obtain unreacted fluorescent label D.
After removing the NA probe and observing the fluorescence generated from the fluorescent substance by irradiating with ultraviolet light, a plurality of fluorescences are observed on the polyoxymethylene needle crystals. Furthermore, if the binding density of the probe is measured, it is 10 ¹¹ to 10 ¹³ molecules / c.
Labeled DNA probes of the order of m ² will be immobilized on the polyoxymethylene needle crystals.

Considering the high aspect ratio of the polyoxymethylene needle-like crystals, the fixation of the DNA probe on the probe-immobilized support produced by the above-mentioned method is less than 1 per unit area as compared with the conventional immobilization on a flat surface.
It is possible to immobilize 0 ² to 10 ³ times as many DNAs.
By using such a probe-immobilized carrier of the present invention, it is possible to significantly improve the sensitivity when detecting a target substance by a measuring method using a fluorescent substance or an electric measuring method.

In the above two examples, the labeled probe was used to show that the probe was immobilized at a high density on the probe-immobilized support. However, in the actual use, labeling was not performed. Not necessarily required.

Although needle-like crystals of polyoxymethylene were described in this example, needle-like crystals of epoxy resin, needle-like crystals of unsaturated polyester resin, thiazyl whiskers, diacetylene-based polymer whiskers, m-chloronitrobenzene. Similarly, the linear, fibrous and columnar structures of organic substances such as whiskers can be solid-phased with the DNA probe.

Example 11. PC using fixed probe support
An example of conditions for a PCR reaction for PCR amplification of a target nucleic acid using the DNA probe provided on the probe-immobilized support described in R reaction examples 1 to 11 as a template is described below. The PCR reaction can be performed under the conditions according to Table 1. That is, a denaturation step at 95 ° C. for 5 minutes, followed by an annealing step at 50 ° C. for 2 minutes and a primer extension step at 72 ° C. for 2 minutes, which are repeated an arbitrary number of times n (where n is an integer of 1 or more). A denaturing step of 2 minutes at 50 ° C., an annealing step of 2 minutes at 50 ° C. following the optional repetition,
Further, the subsequent primer extension step at 72 ° C. for 9 minutes can be performed.

[0341]

[Table 1]

The reaction solutions used in the PCR reaction are summarized in Table 2.

[0343]

[Table 2]

PCR is performed as follows. That is, the probe-immobilized support according to the present invention is directly placed on the heater, the PCR reaction solution having the composition shown in Table 2 is dropped on the DNA chip, and PCR is performed in the reaction step shown in Table 1. In conventional PCR using a glass plate solid phase simple substance, the thermal conductivity is quartz: 1.38 W / mK, Pyrex (registered trademark): 3.99 W / mK at 300 K, so it takes time to reach a predetermined temperature. . However, carbon nanotubes on Si, (Si: 150W / mK) Si
Carbon nanotube on C (SiC: 490W / m
The thermal conductivity of K) is very high, and the temperature can be switched in a short time.

Example 16. Storage Place the probe fixed support prepared in the above step in a clean glass dish and close the lid. This petri dish is stored in a refrigerator in a cool and dark place. Examples 1-above
The life of the DNA chip produced by 11 is
In the case of a probe-immobilized support on which is immobilized for several months, the probe-immobilized support on which oligo DNA is immobilized can be stored for a longer period of time.

Example 17. Probe fixed support for nucleic acid analysis Gene expression analysis can be performed using the probe fixed support of the present invention (generally equivalent to a DNA chip) in which carbon nanotubes are extended on the above-mentioned SiC substrate. Is. Similarly, the probe-immobilized support of the present invention can also be used as a device for carrying out genetic diagnosis on an object to be tested.

In this case, as the probe sequence to be immobilized, a sequence complementary to the sequence of the nucleic acid to be detected may be selected. In the case of gene expression analysis, a probe may be selected for detecting what kind of gene is expressed and / or what kind of gene is not expressed in the test subject. In addition, since the probe-immobilized support of the present invention has excellent quantification, it is possible to analyze changes in the expression level of the gene of interest.

The probe-immobilized support for use in nucleic acid analysis is manufactured by the method described by the examples above. In that case, as the probe to be immobilized, 2 bases to
A nucleic acid fragment having a length of 50 bases, more preferably 10 to 25 bases.
A nucleic acid fragment having a base length is used. The probe is fixed to the probe fixing support by spotting.

For example, nucleic acid fragments are immobilized on the surface of the carbon nanotube layer formed on the SiC substrate in the range of about 10 to about 10 ⁵ types / cm ² , and the fixed ratio of about 10
It is fixed at ¹² molecules / cm ² .

After that, post-treatment with ultraviolet rays, sodium borohydride or Schiff reagent is performed. These post-treatments may be carried out by combining a plurality of types, and it is particularly preferable to carry out a combination of heat treatment and ultraviolet treatment.
It is also preferable to carry out incubation after spotting.
Further, it is preferable to wash and remove the unspotted DNA fragment after the incubation.

An electrochemical method is used as a standard weaving method. This is a method in which an intercalator having a conductive group and having a property of being incorporated into the formed hybrid structure is used to detect the presence or absence of an electric signal via a carbon nanotube having electric conductivity.

A nucleic acid probe having a sequence complementary to a target sequence is immobilized, carbon nanotubes functioning as electrodes are provided as elongated carriers, and the probe is immobilized for electrochemical detection according to the present invention. When an expression analysis is performed using a support, it may be performed as follows.

First, RNA is extracted from an individual by a commercially available kit or a method known per se. C from this RNA
DNA is prepared and used as a sample nucleic acid.

The sample nucleic acid obtained above is added to the probe-immobilized support. Next, a hybridization reaction is performed in the presence of double-stranded recognition and under appropriate conditions.

When the target sequence is present in the sample nucleic acid,
A hybridization reaction occurs, and the double strand thus formed is recognized and bound by the double strand recognizer. At this time, if a potential higher than the potential at which the double-stranded chain-recognizing body reacts electrochemically is applied, the reaction current value derived from the double-stranded chain-recognizing body is measured, and hybridization is detected. Thereby, the presence of the target sequence in the sample nucleic acid is detected.

[0356] For gene diagnosis, probes fixed to one probe-immobilized support are used for analysis of SNPs or mutations of genes associated with a specific disease, and therefore, a plurality of kinds of nucleic acids having different base sequences are used. The probes may be selected as a probe series for specific disease analysis. Alternatively, not limited to a specific disease, a plurality of types of nucleic acid probes having different base sequences may be selected as the individual analysis probe series in order to obtain a large amount of information from a single individual. Further, the probe may be selected so that the information regarding the pathogen contained in the individual can be simultaneously analyzed.

When the DNA chip according to the present invention is used,
Since the specific fixed amount is uniform, not only qualitative analysis based on the presence or absence of hybridization but also quantitative analysis can be performed with high reliability.

[0358]

As described above, according to the present invention, there is provided an apparatus capable of detecting a biological substance with high reliability.

[Brief description of drawings]

FIG. 1 is a diagram showing one embodiment of a probe fixing support according to the present invention.

FIG. 2 is a view showing an example of functional groups introduced into an elongated carrier used in the present invention.

FIG. 3 is a view showing an example of an elongated carrier provided in a probe fixing support according to the present invention.

FIG. 4 is a view showing an example of an elongated carrier provided on a probe fixing support according to the present invention.

FIG. 5 is a view showing an example of an elongated carrier provided on the probe fixing support according to the present invention.

FIG. 6 is a view showing an example of an elongated carrier provided in the probe fixing support according to the present invention.

FIG. 7 is a view showing an example of an elongated carrier provided in the probe fixing support according to the present invention.

FIG. 8 is a view showing an example of an elongated carrier provided in the probe fixing support according to the present invention.

FIG. 9 is a diagram showing a process of manufacturing a probe fixing support according to the present invention from a Si substrate.

FIG. 10 is a diagram showing a Si substrate and carbon nanotubes provided on the Si substrate.

FIG. 11 is a diagram showing a step of manufacturing a probe fixing support according to the present invention from a SiC substrate.

FIG. 12 is a view showing carbon nanotubes provided on a SiC substrate.

FIG. 13 is a diagram showing a step of opening the tip of the carbon nanotube provided on the SiC substrate.

FIG. 14 is a diagram showing a step of chemically modifying the surface of a linear substance of hydroxyapatite.

FIG. 15 is a diagram showing ZnO columnar crystals provided on a (0001) α-Al ₂ O ₃ substrate.

[Explanation of symbols]

1, 31, 60, 70. Probe fixed support 2,3
2,61,71. Substrates 3, 41 ,. Long and thin carrier 4,34,42,63,7
3. Probe 33. Long and narrow carrier assembly 62,7
2. Carbon nanotube 74. Opening 75.
Edge of opening 81. Ni fine particles 82. Board 8
3. Carbon nanotube 84. Probe 10
1. SiC substrate 102, 103. Carbon nanotube 104. Si with carbon nanotubes
C substrate 120. Carbon nanotube 124.
DNA probe 121. SiC substrate 122. Carbon nanotube 123. SiC substrate provided with carbon nanotubes 124. DNA probe
130. Linear substances of hydroxyapatite 13
1. Surface of hydroxyapatite linear material

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/483 C12N 15/00 F 33/551 G01N 27/46 386Z 37/00 102 336G 27/30 351 27 / 46 386G F term (reference) 2G045 DA12 DA13 DA14 DA36 DA37 FB05 FB15 4B024 AA11 CA04 CA09 HA14 4B029 AA07 AA23 FA15

Claims (11)

[Claims] 1. A support, an elongated carrier of an inorganic substance extending through at least one end of the support on one surface thereof, and a probe fixed to the elongated carrier and specifically binding to a target substance. A probe fixing support comprising: 2. The probe fixed support according to claim 1, wherein the elongated carrier is a crystal. 3. The probe fixing support according to claim 1, wherein the elongated carrier is a whisker. 4. The probe fixing support according to claim 1, wherein the elongated carrier is a carbon nanotube. 5. The support is an α-Al ₂ O ₃ single crystal substrate, and the elongated carrier is a ZnO whisker formed by epitaxial growth from the α-Al ₂ O ₃ single crystal substrate. The probe fixing support according to claim 1. 6. The support is a heat-resistant substrate, and the long carriers are carbon nanotubes that are synthesized on the substrate with metal fine particles as nuclei and extend to the surface of the substrate. The probe fixing support according to claim 1. 7. The carbon nanotubes, wherein the support is a substrate having a Si surface and a C surface, are synthesized by heating the substrate in a vacuum, and a plurality of carbon nanotubes are extended to the surface of the substrate, and the carbon nanotubes are respectively extended. A probe-immobilized support comprising a probe that is immobilized one by one and that specifically binds to a target substance. 8. The probe fixing support according to claim 7, wherein the carbon nanotube has an open end. 9. The probe-immobilized support according to claim 1, wherein the target substance is selected from the group consisting of nucleic acids, polypeptides, proteins, antibodies and enzymes. 10. The target substance is a nucleic acid, the probe contains a sequence complementary to the base sequence of the nucleic acid, and the elongated simple substance has electrical conductivity, whereby the probe and the target substance are The probe-immobilized support according to any one of claims 1 to 9, wherein the detection of the bond generated between the two is performed electrochemically. 11. A probe-immobilized carrier comprising an elongated carrier made of an inorganic material and a plurality of probes immobilized on the elongated carrier and specifically binding to a target substance.