US20020146715A1

US20020146715A1 - Ink jet method of spotting probe, probe array and indentification methods

Info

Publication number: US20020146715A1
Application number: US09/951,972
Authority: US
Inventors: Tadashi Okamoto; Nobuko Yamamoto; Tomohiro Suzuki
Original assignee: Individual
Current assignee: Individual
Priority date: 1997-08-01
Filing date: 2001-09-14
Publication date: 2002-10-10
Also published as: US6476215B1; US7994297B2; US20060177868A1; CA2244403C; EP1352976B1; US20030059817A1; EP1352976A2; JPH11187900A; EP0895082B1; ES2206848T3; DE69818371D1; EP0895082A2; CA2244403A1; EP0895082A3; JP4313861B2; ATE250764T1; DK0895082T3; DE69818371T2; EP1352976A3

Abstract

Provided is a method of spotting a probe densely and efficiently on a surface of a solid support. A liquid containing a probe is attached to a solid support as droplets to form spots containing the probe on the solid support by an ink jet method.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of spotting a probe on a solid support, a probe array and a method of manufacturing thereof, and a method of detecting a target single-stranded (ss) nucleic acid and a method of identifying a base sequence of a target ss nucleic acid using the probe array.

2. Related Background Art

As a method to determine a base sequence of a nucleic acid, detect a target nucleic acid in a sample, and identify various bacteria swiftly and accurately, proposed is the use of a probe array where one or more substances which can bind specifically to a target nucleic acid, so-called probes, are arranged on a solid support at a large number of sites. As a general method of manufacturing such probe arrays as described in EP No. 0373203B1, (1) the nucleic acid probe is synthesized on a solid support or (2) a previously synthesized probe is supplied onto a solid support. U.S. Pat. No. 5,405,783 discloses the method (1) in detail. Concerning the method (2), U.S. Pat. No. 5,601,980 and Science Vol. 270, p. 467 (1995) teach a method of arranging cDNA in an array by using a micropipet.

In the above method (1), it is not necessary to synthesize a nucleic acid probe in advance, since the nucleic acid probe is synthesized directly on a solid support. However, it is difficult to purify a probe nucleic acid synthesized on a solid support. The accuracy in determining the base sequence of a nucleic acid and in the detection of a target nucleic acid in a sample using a probe array largely depends on the correctness of the base sequence of the nucleic acid probe. For the method (1), therefore, further improvement in accuracy of a nucleic acid probe is required in order to manufacture a probe array of higher quality. In the method (2), a step of synthesizing a nucleic acid probe is required prior to the fixation of the nucleic acid probe on a solid support, but the nucleic acid probe can be purified before binding the probe to a solid support. For this reason, presently, the method (2) is considered to be more preferable than the method (1) as a method of manufacturing a probe array of high quality. However, the method (2) has a problem in the method of spotting a nucleic acid probe densely on a solid support. For example, when a probe array is used to determine the base sequence of a nucleic acid, it is preferable to arrange as many kinds of nucleic acid probes as possible on a solid support. When mutations in a gene are to be detected efficiently, it is preferable to arrange nucleic acid probes of sequences corresponding to the respective mutations on a solid support. In addition, when a target nucleic acid in a sample or to gene mutations and deletions are detected, it is desirable that the amount of the sample taken from a subject, specifically a blood sample, is as small as possible. Thus, it is preferable that as much information as possible on the base sequence is obtained using a small sample amount. Considering these points, it is preferable that, for example, 10,000 or more nucleic acid probe spots per square inch are arranged in a probe array.

SUMMARY OF THE INVENTION

As the result of the research carried out by the inventors to solve above-discussed problems, they have found that an ink jet ejection method enables spotting of a probe in a markedly high density and achieved the present invention.

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

It is another object of the present invention to provide a probe array that can provide more information on nucleic acid more accurately even using a small amount of sample.

It is still another object of the present invention to provide a method of efficiently manufacturing a probe array, in which a large number of probes are bound to a solid support, without damaging the probes.

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

It is still other object of the present invention to provide a method of identifying the structure of a target substance to obtain information on the structure of the target substance even from a small amount of sample.

According to one aspect of the present invention, there provided is a method of spotting a probe which can bind specifically to a target tot a solid support. The method comprises a step of supplying a liquid containing a probe on a surface of a solid support by an ink jet method and adhering the liquid on the surface of the solid support. The use of the spotting method according to the above embodiment allows accurate and efficient provision of a probe on a solid support and efficient manufacturing of a probe array.

According to another aspect of the present invention, provided is a probe array comprised of a plurality of spots of a probe, where the spots are provided independently at a plurality of sites of the surface of a solid support in a density of 10,000 spots per square inch or higher. This probe array has spots in a remarkably high density so that much information can be obtained even from a small amount of sample.

According to further aspect of the present invention, provided is a method of manufacturing a probe array having a plurality of spots arranged independently in a plurality of sites on a surface of a solid support, the spots containing a probe which can bind specifically to a target substance comprising a step of supplying a liquid containing the probe and attaching the liquid to a predetermined site on the surface of the solid support by means of an ink jet method. According to this embodiment, a probe array comprising spots arranged in a high density can be efficiently manufactured without damaging the probe.

According to further aspect of the present invention, provided is a method of detecting a target substance by contacting a sample with each spot of a probe array having a probe that can bind specifically to a target substance that may be contained in a sample as a plurality of independent spots on a solid support to detect a reaction product of the target substance and the probe on the solid support to detect the presence/absence of the target substance in the sample wherein the respective spots are formed by spotting a liquid containing the probe on the solid support by the ink jet method. According to this embodiment, a target substance can be detected efficiently.

According to further aspect of the present invention, provided is a method of identifying a structure of a target substance contained in a sample comprising:

a step of preparing a probe array provided with spots of a probe, which can bind specifically to a specific substance, on a surface of a solid support;

a step of contacting the sample to the spots; and

a step of detecting binding between the target substance and the probe.

U.S. Pat. No. 5,601,980 states that it is inappropriate to use a conventional ink jet method in spotting of a nucleic acid probe. In lines 31-52 in the second column of U.S. Pat. No. 5,601,980, it is said that the use of the ink jet printer technique in which a small amount of ink is ejected by pressure wave is inappropriate, because the pressure wave for ejecting ink may lead to a drastic rise in the ink temperature and damage the nucleic acid probe and scattering of the ink upon ejection may lead to contamination of adjacent probe spots. Considering this, U.S. Pat. No. 5,601,980 discloses a method of manufacturing a probe array in which a drop of a liquid containing a nucleic acid probe is formed on a tip of a micropipet utilizing gas pressure, while monitoring the size of the drop, application of pressure is terminated when the drop becomes the predetermined size, and the drop is applied on a solid support.

U.S. Pat. No. 5,474,796 discloses manufacturing of oligonucleotide array by forming a matrix of hydrophobic and hydrophilic parts on a solid support surface and ejecting four nucleotides sequentially to the hydrophilic part by means of a piezoelectric impulse jet pump apparatus and a method of determination of the base sequence of a target nucleic acid using the oligonucleotide array. However, these prior arts do not disclose a method in which nucleic acid probes each having a base sequence of a predetermined length is ejected in advance using an ink jet technique to arrange the nucleic acid probes accurately and densely.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view illustrating a method of manufacturing a probe array using a bubble jet head; [0022]
FIG. 2 is a cross sectional view taken along the line [0023] 2-2 of the bubble jet head of FIG. 1;
FIG. 3 shows a graph comparing a theoretical amount and an actual recovery of a nucleic acid probe spotted on an aluminum plate by the bubble jet method in Example 3; [0024]
FIG. 4 shows a graph comparing a theoretical amount and an actual recovery of a nucleic acid probe spotted on an aluminum plate by the bubble jet method in Example 4; [0025]
FIG. 5A is a schematic plan view of one embodiment of a probe array of the present invention, and FIG. 5B is a cross sectional view taken along the [0026] line 5B-5B in FIG. 5A; and
FIG. 6 is to explain a spotting method in Example 8.[0027]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Outline of Method of Manufacturing Probe Array

FIGS. 1 and 2 are schematic diagrams illustrating a method of manufacturing a probe array, for example, a nucleic acid probe array, according to one embodiment of the present invention. In FIG. 1, there are shown a liquid supply system (nozzle) [0028] 101 which ejectably retains a liquid containing a probe, for example, a nucleic acid probe, as an ejection liquid, a solid support 103 (for example, transparent glass plate, etc.) to which the nucleic acid probe is to be bound, and bubble jet head 105, a kind of ink jet heads, provided with a mechanism to apply heat energy to the liquid and thus eject the liquid. 104 denotes a liquid containing a nucleic acid probe ejected from the bubble jet head 105. FIG. 2 is a cross sectional view taken substantially along the line 2-2 of the bubble jet head 105 of FIG. 1. In FIG. 2, there are shown the bubble jet head 105, a liquid 107 containing a nucleic acid probe to be ejected, and a substrate part 117 with a heating member applying ejection energy to the liquid. The substrate part 117 comprises a protective film 109 made of silicone oxide etc., electrodes 111-1 and 111-2 made of aluminum etc., an exothermic resistance layer 113 made of nichrome etc., a heat accumulator layer 115, and a base material 116 made of alumina etc., with good heat radiating properties. A liquid 107 containing a nucleic acid probe comes up to an ejection orifice (ejection outlet) 119 and forms a meniscus 121 by the predetermined pressure. When electric signals from the electrodes 111-1 and 111-2 are supplied, a region shown by 123 (bubbling region) rapidly generates heat and a bubble appears in the liquid 107 contacting the region 123. The meniscus ejects at the pressure and the liquid 107 is ejected from the orifice 119 to fly toward the surface of a solid support 103. Although the ejectable amount of the liquid using a bubble jet head of such a structure depends on the size of the nozzle, etc., it can be controlled to be about 4-50 picoliters and is very useful as means to arrange nucleic acid probes in high density.

Relation between Ejected Liquid and Solid Support

Diameter of Spots on Solid Support [0029]
In order to obtain the probe density as described above (for example, 10,000 probe spots per square inch, upper limit being about 1×10[0030] ⁶) on a solid support, it is preferable that the diameter of each spot is about 20-100 μm, for example, and that spots are independent each other. These spots are determined by properties of a liquid ejected from a bubble jet head and surface properties of the solid support to which the liquid is attached.
Properties of Ejection Liquid [0031]
Any liquid can be used as an ejection liquid, provided that the liquid can be ejected from a bubble jet head, the liquid ejected from the head arrives at the desired positions on a solid support, and the liquid does not damage the nucleic acid probe when it is mixed with nucleic acid probe and it is ejected. [0032]
From a viewpoint of liquid properties to be ejected from a bubble jet head, the liquid preferably has properties such as viscosity of 1-15 cps and surface tension of 30 dyn/cm or higher. When viscosity is 1-5 cps and surface tension is 30-50 dyn/cm, the position of arrival on a solid support becomes significantly accurate and it is especially suitable. [0033]
Then considering ink jet ejection properties of the liquid and stability of a nucleic acid probe in the liquid and, at ejection by a bubble jet, it is preferable to contain a nucleic acid probe of 2-5,000 mer, especially 2-60 mer in a concentration of 0.05-500 μM, especially 2-50 μM. [0034]
Composition of Liquid [0035]
Composition of a liquid to be ejected from a bubble jet head is not particularly restricted, provided that it does not substantially affect a nucleic acid probe when it is mixed with a nucleic acid probe or when it is ejected from the bubble jet head as described above, and a liquid composition normally ejectable to a solid support using the bubble jet head satisfies preferable conditions. However, a preferable liquid contains glycerin, urea, thiodiglycol or ethyleneglycol, isopropyl alcohol, and an acetylene alcohol shown by the following formula (I): [0036]
wherein R1, R2, R3 and R4 represent alkyl groups, specifically straight or branched alkyl groups containing 1-4 carbons, m and n represent integers, and m=0 and n=0 or 1≦m+n≦30, and when m+n=1, m or n is zero. [0037]
More specifically, a liquid comprising 5-10 wt % of urea, 5-10 wt % of glycerin, 5-10 wt % of thiodiglycol, and 0.02-5 wt %, more preferably 0.5-1 wt % of an acetylene alcohol shown by the above formula (I) is preferably used. [0038]
When this liquid is used, spots obtained by ejecting the liquid containing a nucleic acid probe from a bubble jet head and attached on a solid support are round, and an area where the ejected liquid is attached is restricted. Thus, even when a nucleic acid probe is spotted densely, connection of the adjacent spots can be effectively prevented. No degradation of the nucleic acid probe spotted on a solid support is observed. However, the properties of the liquid used in manufacturing a nucleic acid probe array according to the present invention are not restricted to those mentioned above. For example, when structures like wells are provided on a solid support surface to prevent spreading of the liquid applied on the solid support by a bubble jet head and mixing with adjacent spots, a liquid of a viscosity and surface tension out of the above range, and a nucleic acid probe of a base length and concentration out of the above range can be used. [0039]
Kinds of Functional Groups of Solid Support and Nucleic Acids [0040]
A method to securely bind the nucleic acid probe to the solid support, as well as to effectively retain the applied spot of a nucleic acid probe at a more defined position on the solid support to prevent cross contamination between adjacent spots, one can endow the probe and the solid support with functional groups which can react each other. [0041]
SH Group and Maleimido Group [0042]
The combination use of the maleimido group and the thiol (—SH) group can be mentioned as a preferable example. That is, by binding a thiol (—SH) group to the terminus of a nucleic acid probe and treating the solid support surface to have a maleimido group, the thiol group in a nucleic acid probe when supplied to the surface of the solid support reacts with the maleimido group of the solid support to immobilize the nucleic acid probe on the support, forming probe spots on the predetermined positions on the solid support. Especially, when such a nucleic acid probe containing a thiol group at the terminus is dissolved in a liquid of the above-mentioned composition, and applied on a solid support surface having maleimido groups by means of a bubble jet head, the nucleic acid probe solution can form a very small spot on the solid support. As a result, small spots of a nucleic acid probe can be formed on the predetermined positions of the surface of the solid support. In this case, it is not necessary to provide a construction such as wells comprised of partly hydrophilic and hydrophobic matrix on the surface of the solid support to prevent connection between spots. [0043]
For example, when a liquid containing a nucleic acid probe of 18 mer nucleotides at a concentration of 8 μM and controlled to have the viscosity and surface tension within the above ranges was ejected from a nozzle (an amount of ejection about 24 picoliters) using a bubble jet printer (Product name: BJC620; Canon Inc., modified to print on a flat plane) with a space between the solid support and the nozzle tip of the bubble jet head set about 1.2-1.5 mm, spots of a diameter about 70-100 μm could be formed and no spots due to scattering when the ejected liquid hit the surface of the solid support (referred to as satellite spots hereinafter) were observed. Reaction between maleimido groups on the solid support and SH groups at the terminus of the nucleic acid probes is completed in about 30 minutes at room temperature (25° C.), although depending on the conditions of an ejected liquid. The time required is shorter than that required when a piezoelectric jet head is used to eject a liquid. Although the reason is not known, it is considered that the temperature of the nucleic acid probe solution in the bubble jet head is elevated according to its base principle so that the efficiency of reaction between a maleimido group and a thiol group is increased to shorten the reaction time. [0044]
Incidentally, a thiol group tends to become unstable under an alkaline or neutral conditions and a disulfide bond (—S—S—), which gives a dimer, may be formed. In order to prevent the disulfide bond formation and to accomplish an effective reaction between a thiol group and a maleimido group, it is preferable to add thiodiglycol to the ejection liquid. [0045]
In order to introduce maleimido group onto a solid support surface, various methods can be employed. For example, when a glass substrate is used as the solid support, maleimido group can be incorporated onto the surface of the solid support by an introduction of amino group onto the substrate and the following reaction between the amino group and a reagent containing N-(6-maleimidocaproyloxy)succinimide (EMCS reagent: Dojin Co., Ltd.). The amino group introduction onto the surface can be conducted by reacting an aminosilane coupling agent with the glass substrate. [0046]
Structural Formula of EMCS [0047]
A nucleic acid probe having a thiol group at the terminus thereof can be obtained by synthesizing a nucleic acid using 5′-thiol-modifier C6 (Glen Research Co., Ltd.) as a reagent for the 5′-terminus on an automatic DNA synthesizer followed by usual deprotection reaction and purification by high performance liquid chromatography. [0048]
Amino Group and Epoxy Group [0049]
As functional groups used for immobilization other than the above-mentioned combination of the thiol group and the maleimido group, a combination of the epoxy group (on solid support) and the amino group (nucleic acid probe terminus) may also be used. Epoxy groups can be introduced onto a solid support surface, for example, by applying polyglycidyl methacrylate having an epoxy group onto the surface of a solid support of a resin, or by applying a silane coupling agent having an epoxy group onto the surface of glass solid support for reaction. [0050]
As explained above, when functional groups are introduced into a solid support surface and a terminus of a ss-nucleic acid probe to form covalent bonds, the nucleic acid probe is more firmly fixed to the solid support. In addition, since the nucleic acid probe always binds to the solid support at its terminus, the states of the nucleic acid probe in each spot become homogeneous. As a result, hybridization between the nucleic acid probes and target nucleic acids occurs in uniform conditions, thus the detection of a target nucleic acid and the identification of a base sequence with further improved accuracy can be realized. When nucleic acid probes having a functional group on each terminus are covalently bound to a solid support, a probe array can be produced quantitatively without differences in the amount of bound probe DNA due to difference in sequence or length, compared with nucleic acid probes fixed on a solid support by non-covalent bond (for example, electrostatically, etc.). In addition, all parts of the nucleic acid participate in hybridization reaction, efficiency of hybridization can be markedly improved. In addition, a linker such as alkylene groups of 1-7 carbons or ethylene glycol derivatives can be present between the ss nucleic acid portion which hybridizes with a target nucleic acid and the functional group for binding with a solid support. When such a nucleic acid probe is bound to a solid support, a predetermined space can be provided between the surface of the solid support and the nucleic acid probe so that efficacy of reaction between a nucleic acid probe and a target nucleic acid can be expected to be improved further. [0051]
Manufacturing Method of Probe [0052]
One of the preferred embodiments of the probe array-manufacturing method will now be explained. First, a liquid containing 7.5 wt % of glycerin, 7.5 wt % of urea, 7.5% of thiodiglycol, and 1 wt % of an acetylene alcohol shown by the above general formula (I) (for example, Product Name: Acetylenol EH; Kawaken Fine Chemical Co., Ltd.) is prepared. A ss nucleic acid probe of a length of, for example, about 2-5,000 mer, especially, about 2-60 mer, having a thiol group at the terminus is synthesized using an automatic DNA synthesizer. Nest, the nucleic acid probe is mixed in the above liquid at a concentration in a range of 0.05-500 μM, especially 2-50 μM, to produce a liquid to be ejected having a viscosity of 1-15 cps, especially 1-5 cps, and surface tension of 30 dyn/cm or higher, especially 30-50 dyn/cm. Then, this ejection liquid is filled in a nozzle of a bubble jet head, for example. Maleimido groups are introduced on a solid support surface according to the above method. The solid support is placed so that a distance between the surface of the solid support having maleimido groups and the nozzle tip of the bubble jet head becomes as close as about 1.2-1.5 mm, and the bubble jet head is driven to eject the liquid. Here, as the ejection conditions, it is desirable to set printing pattern so as not to allow the connection between the spots on a solid support each other. When a bubble jet head of which resolution is 360×720 dpi is used for spotting, preferable conditions are that one liquid ejection is followed by twice idle ejections in the 360 dpi direction and one liquid ejection is followed by 5 times idle ejections in the 720 dpi direction. These conditions can provide a space of about 100 μm between spots and sufficiently prevent contamination between adjacent spots. Then, the solid support is stood, for example, in a humid chamber, until a reaction between the maleimido groups on a solid support and the thiol groups of nucleic acid probes in a liquid proceeds and the nucleic acid probes are securely fixed on the solid support. It is sufficient to leave it at room temperature (about 25° C.) for about 30 minutes as described above. Then, the nucleic acid probes not reacted on the solid support are washed away to obtain a nucleic acid probe array. [0053]
Now, in order to improve detection accuracy (S/N ratio) in, for example, detection of a target nucleic acid using this nucleic acid probe array, it is preferable to block the solid support surface after the nucleic acid probes were fixed to the support to prevent the surface areas not binding the nucleic acid probes from reacting with a target nucleic acid, etc., contained in a sample. Blocking can be performed by, for example, immersing the solid support in a 2% aqueous bovine serum albumin solution for two hours or decomposing maleimido groups not bound to the nucleic acid probes on the surface of the solid support. For example, DTT (dithiothreitol), β-mercaptoethanol, etc. can be used. However, in terms of an effect of preventing adsorption of target DNA, an aqueous solution of bovine serum albumin is the most suitable. This step of blocking may be performed, as required. For example, this blocking step can be omitted, when a sample can be supplied restrictively to the respective spots of the probe array and any sample would not attach substantially to the parts other than the probe spots. The blocking step can be omitted, also when wells have been formed on the solid support beforehand, and parts other than wells are processed to inhibit attachment of nucleic acid probes. [0054]
The probe arrays manufactured by such a method may have a plurality of spots containing the same nucleic acid probe or a plurality of spots each containing a different nucleic acid probe, depending on applications. The probe array in which the nucleic acid probes are arranged at a high density prepared a mentioned above, can then be used for the detection of a target ss nucleic acid and the identification of a base sequence. For example, when a target ss nucleic acid of a known base sequence which may be present in a test sample is detected, a ss nucleic acid having a base sequence complementary to that of the target nucleic acid is used as the probe, and the probe array in which a plurality of spots containing the probe are arranged on a solid support is prepared. Each sample is supplied to each spot of the probe array, and the probe array is left standing under conditions allowing hybridization between the target nucleic acid and the probe, then the presence/absence of hybrid in each spot is detected by a known method such as fluorescent detection. This enables detection of the presence/absence of the target substance in a sample. When a probe array is used to identify a base sequence of a target ss nucleic acid contained in a sample, a plurality of ss nucleic acids having base sequences complementary to the presumed sequences of the target nucleic acid are spotted as probes on the solid support. Then, aliquots of the sample are supplied to the respective spots and incubated under conditions allowing hybridization of the target nucleic acid and the probe, and then the presence/absence of hybridization at each spot is detected by a known method such as the fluorescence method. This enables identification of a base sequence of a target ss nucleic acid. As other applications of the probe array according to the present invention, for example, application to screening of specific base sequences recognized by DNA binding protein and chemical substances having a property to bind to DNA can be considered. [0055]
Kinds of Ink Jet Head [0056]
Although a constitution in which a nucleic acid probe is applied to a solid support by means of a bubble jet head is solely illustrated above, a piezoelectric jet head ejecting a liquid in a nozzle by vibration pressure of piezoelectric elements can also be used in the present invention. However, a bubble jet head is suitably used in the present invention, since a binding reaction to a solid support is completed in a short period of time and secondary structure of DNA is unfolded by heat so that efficiency of the subsequent hybridization reaction can be increased, as described above. [0057]
In addition, an ink jet system having a plurality of heads can be used to form a plurality of spots simultaneously on a solid support so that two or more spots may contain different nucleic acid probes. [0058]
PNA/DNA [0059]
The present invention has been illustrated using a nucleic acid probe as an example of probes. Nucleic acid probes include deoxyribonucleic acid (DNA) probes, ribonucleic acid (RNA) probes, and peptide nucleic acid (PNA) probes. PNAs are synthetic oligonucleotides in which four bases (adenine, guanine, thymine, and cytosine) contained in DNA are bound to a peptide backbone, not to a sugar-phosphate backbone as shown in the following formula (II): PNA Structural Formula (II) [0060]
wherein “Base” represents any one of four bases (adenine, guanine, thymine, and cytosine) contained in DNA, and p represents a base length of the PNA. PNAs can be synthesized, for example, by methods known as tBOC-type solid phase synthesis and Fmoc-type solid phase synthesis. PNAs are more resistant to enzymes such as nucleases and proteases as compared to natural oligonucleotides of DNA and RNA, hardly or not cleaved enzymatically, and stable in the serum. Due to the absence of the sugar moiety or phosphate groups, PNAs are rarely affected by ionic strength of a buffer. Therefore, it is not required to control a salt concentration, etc., when PNAs are reacted with a target ss nucleic acid. In addition, due to the absence of electrostatic repulsion, a hybrid between PNA and a target ss nucleic acid is considered to be more heat-stable than those between a DNA probe and a target ss nucleic acid and between an RNA probe and a target ss nucleic acid. From these characteristics, PNAs are expected as probes used for the detection of a target nucleic acid and the determination of a base sequence. The method of manufacturing a nucleic acid probe array according to the present invention is effective also when a PNA probe is used as a nucleic acid probe and can easily manufacture a PNA probe array in which PNA probes are arranged densely and very accurately. Specifically, for example, to increase the density of a probe array by securing a PNA probe on restricted positions on a solid support, as in the case of DNA probes and RNA probes, two kinds of functional groups which can react each other into the terminus of a PNA probe and a solid support surface are introduced respectively. A preferred combination of reactive groups is, as mentioned above, a combination of a thiol group (at the terminus of PNA) and a maleimido group (a solid support surface). A thiol group can be introduced at the terminus of PNA by, for example, introducing a cysteine (CH(NH[0061] ₂)(COOH)CH₂SH) group, etc., containing a thiol group in the N-terminus (corresponding to the 5′-terminus of DNA) of a PNA probe. A cysteine group can be introduced at the N-terminus of a PNA probe by, for example, reacting the amino group of the N-terminus of a PNA probe and the carboxyl group of cysteine. Further, using a suitable linker such as those containing an amino group and a carboxyl group such as N₂H(CH₂)₂O(CH₂)₂OCH₂COOH, the amino group at N-terminus of a PNA probe is reacted with the carboxyl group of the linker and then the amino group of the linker is reacted with the carboxyl group of cysteine so as to bind cysteine to the PNA probe via the linker. When a binding group to a solid support is introduced via a linker as mentioned above, a part of PNA probe interactive with a target substance can be separated from the solid support by a predetermined distance so that a further improvement in hybridization efficiency is expected.
PNA may have lower water-solubility than DNA of the same base length as the polymer length of the PNA. Thus, when a liquid for ink jet ejection is prepared, it is preferable to dissolve PNA in trifluoroacetic acid (for example, a 0.1 wt % aqueous solution of trifluoroacetic acid) etc., in advance and then prepare an ejection liquid of properties compatible to ink jet ejection using various solvents mentioned above. In particular, prior dissolution in trifluoroacetic acid can prevent the conversion of the terminal cysteine residues to cystine due to the oxidation of thiol groups of PNA. Thus it is preferable for further improvement in efficiency of a reaction between the thiol group of PNA and the maleimido group on a solid support surface. Although the reaction time of 30 min is sufficient for a reaction between a thiol group introduced at the terminus of a DNA probe or an RNA probe and a maleimido group on a solid support surface (when a bubble jet head is used), it is preferable to proceed a reaction for about 2 hours in case of PNA even using a bubble jet head. [0062]
In the present invention, probes are not limited to nucleic acid probes, and include substances which can bind specifically to a target substance in a sample to be detected or analyzed, for example, ligands which can bind specifically to receptors, receptors which can bind specifically to ligands, oligopeptides and polypeptides which can bind to oligopeptides and polypeptides having specific amino acid sequences, and proteins (for example, antibodies, antigens, enzymes, etc.). [0063]
As mentioned above, according to the method of manufacturing a probe array comprising a step of supplying a probe solution to a solid support using an ink jet ejection process, a probe array can be manufactured very easily. In particular, when functional groups are introduced both in a nucleic acid probe and in a solid support surface so as to form a covalent bond between them, adjacent spots do not connect each other even when a solid support on which wells, etc. have not been provided in advance, that is, a solid support which is substantially flat and has homogenous surface properties (water-wettability, etc.) is used. As a result, a nucleic acid probe array in which spots of a nucleic acid probe are arranged accurately and densely can be manufactured extremely efficiently and at a low cost. [0064]
This description does not intend to exclude a solid support provided with wells on the surface in the present invention. For example, when opaque matrix pattern (referred to as a black (BM) matrix hereinafter) is previously formed between wells to which a probe solution is supplied, detection accuracy (SN ratio) can be further improved in optical detection (for example, detection of fluorescence) of hybridization between a probe and a target substance. In addition, when a matrix whose surface has a low affinity to a probe solution is provided between adjacent wells, the probe solution can be smoothly supplied to desired wells, even when the solution is supplied to somewhat offset positions during supply of the probe solution to wells. To enjoy such an effect, it can be used a solid support on the surface of which wells are provided. A solid support with a matrix formed on its surface, a manufacturing method thereof, and a method of using the solid support according to this embodiment are described below. [0065]
FIG. 5A and 5B show examples of a probe array according to this embodiment of the present invention. FIG. 5A is a plan view and FIG. 5B is a cross sectional view taken along the [0066] line 5B-5B of FIG. 5A. This probe array has a configuration in which a matrix pattern 125 in a framework structure containing hollowed parts (wells) 127 are arranged in a form of a matrix are formed on a solid support 103. The wells 127 separated by the matrix pattern 125 (projecting part) are provided as through holes (bored parts) in the matrix pattern, the side walls of the holes being formed by projecting parts, and a surface of the solid support 103 is exposed at the bottom 129. The exposed surface of the solid support 103 forms a surface which can bind to a probe, and probes (not shown) are fixed to the predetermined wells.
Materials to form the matrix pattern are preferably those which make the matrix pattern opaque, considering improvement in detection sensitivity, S/N ratio, and reliability, when a reaction product of a probe and a target substance is detected optically, for example, by measuring florescence emitted from the reaction product. As these materials, metals (chromium, aluminum, gold, etc.) and black resins, etc., can be exemplified. As the black resins, included are resins such as acrylic, polycarbonate, polystyrene, polyethylene, polyimide, acrylic monomer, and urethane acrylate and photosensitive resins such as photoresists containing black dyes or pigments. As specific examples of photosensitive resins, for example, UV resist, DEEP-UV resist, ultraviolet cure resins can be used. As UV resists, negative resists such as cyclized polyisoprene-aromatic bisazide resists, and phenol resin-aromatic azide compound resists, and positive resists such as novolak resin-diazonaphtoquinone resists can be mentioned. As DEEP-UV resists, positive resists, for example, radiolytic polymer resists such as polymethyl methacrylate, polymethylene sulfone, polyhexafluorobutyl methacrylate, polymethylisopropenyl ketone, and poly-1-trimethylsilyl propylene bromide and dissolution suppressant resists such as o-nitrobenzyl ester cholate, and negative resists such as polyvinylphenol-3,3′-diazidediphenyl sulfone and glycidyl polymethacrylate can be mentioned. [0067]
As ultraviolet curing resins, polyester acrylate, epoxy acrylate and urethane acrylate, etc., containing about 2-10 wt % of one or more photopolymerization initiators selected from a group consisting of benzophenone and its substituted derivatives, benzoin and its substituted derivatives, acetophenone and its substituted derivatives, and oxime compounds such benzyl, etc can be mentioned. [0068]
As black pigments, carbon black and black organic pigments can be used. [0069]
When the reaction product of a probe and a target substance is not detected optically and when light from a matrix does not affect optical detection of a reaction product, the use of non-light-shielding substances as a material for a matrix pattern is not excluded. [0070]
As one of the methods of forming a matrix pattern using the above materials, a method in which a photoresist layer is formed on a resin or a metal layer formed on the surface of a substrate, and after the patterning of the resist layer, the resin is patterned by a process such as etching. When a photosensitive resin is used, the resin itself can be exposed, developed, and cured if required, by a process of photolithography using a photomask for patterning. When a [0071] matrix 125 is made of a resin, the surface of the matrix 125 is hydrophobic. This configuration is preferable when an aqueous solution is used as a solution containing a probe and supplied to wells. That is, when a probe solution is supplied to wells by the ink jet method, the probe solution can be supplied very smoothly to desired wells, even when the probe solution is supplied in slightly offset positions. In addition, when different probes are supplied to adjacent wells simultaneously, cross-contamination of these different probe solutions supplied to the wells can be prevented.
Since a solution of a probe, a biomaterial, such as peptides and nucleic acids, is often an aqueous solution, this constitution in which a matrix pattern is water-repellent can be suitably used in such occasions. [0072]
Next, a method of making a bottom of a well (an exposed part of a solid support surface) which can bind a probe is described. A functional group to be retained on the bottom of a well is determined by the functional group to be carried on a probe. For example, when a nucleic acid probe in which a thiol group is introduced at the terminus is used, previous introduction of a maleimido group to a solid support surface, as mentioned above, makes the thiol group of the nucleic acid probe supplied to wells form a covalent bond with the maleimido group on the surface of the solid support and the nucleic acid probe is then fixed on the surface of the solid support. Similarly, with a nucleic acid probe having an amino group at the terminus, it is preferable to introduce epoxy groups to a solid support surface. As other combinations of these functional groups, for example, a combination of a carboxyl group for a nucleic acid probe (by introducing a succinimide derivative to the terminus of a nucleic acid probe) and an amino group for a solid support surface is preferable. This combination of amino and epoxy groups is inferior in immobilization of the ink jet-ejected nucleic acid probe on a solid support to a combination of thiol and maleimido groups but to a negligible extent when wells are provided on the solid support. [0073]
The amino or epoxy group can be introduced to a glass plate as the solid support by, first treating the surface of the glass plate with an alkali solution such as potassium hydroxide and sodium hydroxide to expose hydroxyl groups (silanol groups) to the surface, and then reacting a silane coupling agent containing a silane compound to which an amino group has been introduced (for example, N-β-(aminoethyl)-γ-aminopropyltrimethoxysilane, etc.) or a silane compound to which an epoxy group has been introduced (for example, γ-glycidoxypropyltrimethoxysilane, etc.) with a hydroxyl group of the surface of the glass plate. To introduce maleimido groups to the surface of the glass plate, the amino groups introduced by the above method are reacted with N-maleimidocaproyloxy succinimide or succinimidyl-4-(maleimido phenyl)butyrate, etc. [0074]
The structures of N-β-(aminoethyl)-γ-aminopropyltrimethoxysilane, γ-glycidoxypropyltrimethoxysilane, and succinimidyl-4-(maleimido phenyl)butyrate are shown below: [0075]
N-β-(aminoethyl)-γ-aminopropyltrimethoxysilane (CH[0076] ₃O)₂SiC₃H₆NHC₂H₄NH₂
γ-glycidoxypropyltrimethoxysilane [0077]
(CH[0078] ₃O)₃SiC₃H₆OCH₂
Succinimidyl-4-(maleimido phenyl)butyrate [0079]
When an epoxy group is introduced to a solid support surface in the above surface treatment of a solid support, the base of wells can be made hydrophilic after binding the epoxy groups to a probe, by opening unreacted epoxy rings using an aqueous solution of ethanol amine, etc., to change them into hydroxyl groups. This operation is preferable, when an aqueous solvent containing a target substance that will react specifically to a probe is supplied to wells to which the probe has been bound. [0080]
When a resin plate is used as a solid support, hydroxyl groups, carboxyl groups, or amino groups can be introduced to the surface of resin substrate according to the method described in Chapter 5 of “Organic Thin Films and Surface”, Vol. 20, Academic Press. Alternatively, after introducing hydroxyl groups by this method, as is shown for the glass plate mentioned above, amino groups or epoxy groups can be introduced by using a silane compound having amino group or epoxy group. Further a maleimido group can be introduced. Functional groups can be introduced either before or after the matrix pattern is formed on a solid support. Before matrix pattern formation, a reaction solution required for introduction of a functional group can be supplied to a solid support surface by spin coating or dip coating, etc. After matrix formation, a reaction solution can be supplied to each well by the ink jet method, etc. [0081]
To bind a probe to a resin substrate, for example, hydroxyl groups are introduced by oxidation of the surface of a resin substrate, then the hydroxyl groups are reacted with a silane coupling agent comprised of a silane compound containing an amino group to introduce amino groups, and each amino group is reacted with a functional group of a probe, as described in Japanese Patent Application Laid-Open No. 60-015560. [0082]
When the substrate after treatment is hydrophilic, above-mentioned resins to make matrix pattern formation can be used without any treatment as a relatively water-repellent material. When further repellency is required, a water-repellant can be added to a matrix material. When a matrix pattern is formed from a photosensitive resin such as photoresists, post-baking under appropriate conditions following exposure and development can provide stronger repellency to the matrix pattern. [0083]
When a probe solution is lipophilic, although it has been explained mainly on a hydrophilic probe solution, treatment can be performed in opposite. [0084]
The size and shape of wells in a matrix pattern can be selected according to the size of a substrate, the size of an array as a whole finally prepared, the number and a type of probe constituting the array, or a method of forming a matrix pattern, a method of supplying a probe solution to wells in matrix pattern, and a method of detection, etc. [0085]
Cross section of wells by a plane parallel to the substrate can be various shapes, in addition to squares as shown in FIG. 5, such as rectangles, various polygons, circles, and ovals. [0086]
Preferably, wells have a maximum width of 300 μm or less, considering the number of reactants and a size of a whole array. For example, as shown in FIG. 5, when a cross section taken parallel to a substrate is square, one side can be 200 μm or less in length. Preferably, when wells are rectangular, the maximum side is 200 μm or less, and when wells are round, the diameter is 200 μm or less. The minimum limit in length is about 1 μm. [0087]
Wells can be arranged in various patterns as required. Wells can be arranged at equal intervals making rows and columns as shown in FIG. 5, or wells can be arranged so as to shift from the positions of wells in adjacent lines. [0088]
A distance between adjacent wells is preferably set not to cause cross-contamination even when the ejection positions are somewhat offset from the position of the target well to which a probe solution is supplied by, for example, the ink jet method. In addition, considering a size of a whole array, cross-contamination, and handling properties in supply of various solutions, the distance between the adjacent wells is in the range of 1/2 to 2 times the maximum width. [0089]
For example, it is desirable 100×100 or 1,000×1,000 or more types of probes are present in a probe array for fully displaying functions of combinatorial chemistry, and the size of a substrate is desirably 1 inch×1 inch or 1 cm×1 cm, to be suitable for automation of operations such as probe fixation, sample supply and detection, thus for square wells it is preferable to set a side of a square of a well at 1-200 μm or less and a distance between adjacent wells is at 200 μm or less, considering the matrix size. [0090]
The thickness of a matrix (height from the solid support surface) is determined considering a method of forming the matrix pattern, volume of wells, and volume of a probe solution supplied. It is preferably 1-20 μm. Such a thickness enables, when a probe solution is supplied to each well by the ink jet ejection method, to retain the probe solution at predetermined positions on a solid support and to prevent cross-contamination very efficiently, even when the properties of the probe solution should be not suitable for forming small spots on the solid support surface, in relation to the conditions for the ink jet ejection method. [0091]
When a well has a size of the upper limits of the above-described desirable ranges, that is, 200 μm×200 μm×20 μm, the well volume is 800 pl. When this size is used and a distance between adjacent wells (x in FIG. 1) is also set at 200 μm, a density of wells of 625 wells/cm[0092] ²is obtained. That is, an array with a well density of an order of 10²wells/cm²or more can be obtained. When a well is a square with a side of 5 μm, a distance between adjacent wells is set at 5 μm, and a thickness of the matrix pattern is set at 4 μm, a volume of a well is 0.1 pl and the density of wells is 1,000,000 wells/cm². Since patterning of 5 μm×5 μm×4 μm is possible in the present fine processing technology, an array with a well density of an order of 106 wells/cm²or more can be included in the scope of the present invention.
In this embodiment, the feeding volume of a probe solution or a substance to be reacted with a probe supplied to a well is 0.1 picoliters (pl) to 1 nanoliter (nl) from the above calculation, when the volume to be supplied is deemed to be the same as or almost the same as the volume of the well. When a matrix has little affinity to a solution to be supplied, it is possible to supply the solution in an volume exceeding the well volume which is retained above the opening of the well due to surface tension, depending to the type of the solution. In such a case, for example, a solution in a [0093] volume 10 to several tens of times larger than that of the well can be supplied and retained. That is, several picoliters to several tens of nanoliters of a solution are supplied. In any cases, a probe solution is preferably supplied to wells using the ink jet method that can supply such a small amount of solution with position accuracy and supply accuracy, although microdispensers and micropipettes can also be used. In the ink jet printing, an ink is ejected with positioning at high accuracy of an order of μm. This method is thus quite suitable for supplying a solution to wells. Since a volume of ink to be ejected is several tens of picoliters to several nanoliters, the ink jet method can be said to be suitable for supplying a solution, also in this respect.
According to this embodiment, spreading of droplets can be controlled quantitatively by the reaction between a nucleic acid probe and a solid support surface as well as by wells. In addition, even when a liquid is ejected in a somewhat offset direction, when a droplet lands on an area containing a well, the droplet part on the matrix is repelled and drawn into the well smoothly, since the matrix has no affinity to the ejected solution. [0094]
The ink jet method used in the present invention is not particularly restricted, and a piezo jet method, a bubble jet method utilizing thermal bubbling, etc., can be employed. [0095]
Any materials can be used as the [0096] solid support 103 according to one embodiment of the present invention, so long as various functional groups as described above can be introduced to the surface. According to the second embodiment of the present invention, preferred materials are those on the surface of which a matrix pattern can be formed. When the reaction product of a probe and a target substance is detected optically by a detection system via a solid support, the solid support is preferably transparent. As these materials, glass including synthetic quartz and fused quartz, silicone, acrylic resins, polycarbonate resins, polystyrene resins, and vinyl chloride resins, etc. can be mentioned. When the reaction product is detected optically not via a solid support, it is preferable to use an optically black solid support, and resin substrates containing black dyes or pigments such as carbon black are used.
In the present invention, a solution which may contain a substance which reacts with the probe (a test solution) is supplied to a probe array and left under suitable reaction conditions to proceed the reaction. When plural test solutions must be supplied to the array, at least one test solution is supplied to plural wells in the probe array, respectively. In this case, as shown above, when the supplied solution has an affinity to wells containing a fixed probe in the already formed probe array and has no affinity to a matrix pattern, quantitative supply of the solution to a restricted supply area can be achieved without cross-contamination. Since most of biomaterials are water-soluble, wells are hydrophilic and a matrix pattern is water-repellent. In addition, the use of the ink jet method in supply of these substances for reaction as shown above can quantitatively supply a very small amount of solution. [0097]
According to the present invention, very small amounts of a probe solution and a test solution are used. Thus, it is desirable to include conditions for preventing evaporation or vaporization of the supplied solutions for both cases. [0098]
The present invention is described in more detail referring to the following examples. [0099]

EXAMPLE 1

Manufacturing of Nucleic Acid Probe Array Using Bubble Jet Printer and Evaluation of the Probe Array [0100]
(1) Washing of Substrate [0101]
A glass plate of 1 inch×1 inch was placed in a rack and immersed in an ultrasonic washing detergent overnight. After ultrasonic washing in the detergent for 20 minutes, the detergent was removed by rinsing with water. After rinsing with distilled water, ultrasonic treatment was performed in a container containing distilled water for 20 minutes. The glass plate was immersed for 10 minutes in a 1 N sodium hydroxide solution preheated to 80° C. Then, the plate was washed with water and distilled water to prepare a glass plate for a probe array. [0102]
(2) Surface Treatment [0103]
A 1 wt % aqueous solution of a silane coupling agent (Product name: KBM603; Shin-Etsu Chemical Co., Ltd.) containing a silane compound having an amino group (N-β-(aminoethyl)-γ-aminopropyltrimethoxysilane) was stirred at room temperature for 2 hours to hydrolyze methoxy groups of the above silane compound. Then, the substrate was immersed in this solution at room temperature (25° C.) for 20 minutes, drawn up from the solution, and dried by blowing nitrogen gas to both sides of the glass plate. Then, the glass plate was baked for 1 hour in an oven heated to 120° C. to complete silane coupling treatment to introduce an amino group on the surface of the substrate. Then, 2.7 mg of N-(6-maleimidocaproyloxy) succinimide (Dojin Co., Ltd.) (abbreviated as EMCS hereinafter) was weighed and dissolved in a mixture of DMSO/ethanol (1:1) to a final concentration of 0.3 mg/ml to prepare an EMCS solution. The glass plate subjected to silane coupling treatment was immersed in the EMCS solution at room temperature for 2 hours for the reaction of the amino groups carried on the surface of the glass plate by silane coupling treatment and the carboxyl groups of the EMCS solution. In this condition, the glass plate obtained maleimido groups derived from EMCS on its surface. The glass plate drawn up from the EMCS solution was washed successively with a mixed solvent of dimethylsulfoxide and ethanol and with ethanol and then dried under a nitrogen gas atmosphere. [0104]
(3) Synthesis of DNA Probe [0105]
A single-stranded (ss) nucleic acid of SEQ ID No. 1 was synthesized using an automatic DNA synthesizer. A thiol (—SH) group was introduced at the terminus of the ss DNA of SEQ ID No. 1 using Thiol-Modifier (Glen Research Co., Ltd.) during synthesis by the automatic DNA synthesizer. Following ordinary deprotection, DNA was recovered, purified with high performance liquid chromatography, and used in the following experiments. [0106]
SEQ ID No. 1 [0107]
[0108] ^5′HS—(CH₂)₆—O—PO₂—O-ACTGGCCGTCGTTTTACA ^3′
(4) DNA Ejection and Binding to Substrate Using BJ Printer [0109]
The ssDNA of SEQ ID No. 1 was dissolved in a TE solution (10 mM Tris-HCl (pH 8)/1 mM EDTA aqueous solution) to a final concentration of about 400 mg/ml to prepare a ssDNA solution (accurate concentration is calculated from absorbance). [0110]
An aqueous solution containing glycerin at 7.5 wt %, urea at 7.5 wt %, thiodiglycol at 7.5 wt %, and an acetylene alcohol (Product name: Acetylenol EH; Kawaken Fine Chemical Co., Ltd.) having the above general formula (I) at 1 wt % was prepared and added to the DNA solution to adjust a final concentration of the ssDNA to 8 μM. This liquid had surface tension in a range of 30-50 dyn/cm and viscosity of 1.8 cps (E-type viscometer: Tokyo Keiki Co., Ltd.). This liquid was filled in an ink tank of a bubble jet printer (Product name: BJC620; Canon Inc.) and the ink tank was mounted on a bubble jet head. The bubble jet printer used here (Product name: BJC620; Canon Inc.) had been modified to enable printing on a plate. This bubble jet printer can print with a resolution of 360×720 dpi. The glass plate treated in the above (2) was then mounted on this printer and the liquid containing the probe nucleic acid was spotted on the glass plate. The distance between the nozzle tip of the bubble jet head and the surface of the glass plate was 1.2-1.5 mm. The conditions for spotting were set in such a manner that the liquid was spotted once followed by 2 idle ejections in a direction of 360 dpi and then spotted once followed by 5 idle ejections in a direction of 720 dpi. After completion of spotting, the glass plate was left to stand in a humid chamber for 30 minutes to complete the reaction between the maleimido groups on the glass plate surface and the thiol groups at the terminus of the nucleic acid probes. The amount of the DNA solution ejected by one ejection operation of the printer was about 24 pl. [0111]
(5) Blocking Reaction [0112]
After completion of the reaction between the maleimido group and the thiol group, the glass plate was washed with an 1 [0113] M NaCl 50 mM phosphate buffer solution (pH 7.0) to rinse completely away the liquid containing DNA on the surface of the glass plate. Then, the glass plate was immersed in a 2% bovine serum albumin aqueous solution and left for 2 hours to proceed a blocking reaction.
(6) Hybridization Reaction [0114]
A ssDNA with a base sequence complementary to DNA of SEQ ID No. 1 was synthesized using an automatic DNA synthesizer, and rhodamine was bound to its 5′-terminus to obtain a labeled ssDNA. This labeled ssDNA was dissolved in 1 M NaCl/50 mM phosphate buffer solution (pH 7.0) to a final concentration of 1 μM. The probe array subjected to the blocking treatment obtained in the above (5) was immersed in this solution at room temperature (25° C.) for 3 hours to proceed a hybridization reaction. Then, the probe array was washed with 1 M NaCl/50 mM phosphate buffer solution (pH 7.0) to wash away the ssDNA which had not been hybridized with the probe nucleic acid. Then, the fluorescence intensity of each spot of the probe array was quantified using the image analyzer (Product name: ARGUS; Hamamatsu Photonics Co., Ltd.). [0115]
(7) Results [0116]
The fluorescence intensity of the spots of the nucleic acid of SEQ ID No. 1 completely matched with the labeled ssDNA was 4,600. In addition, the probe array in which the respective spots emitted fluorescence after hybridization was observed using a fluorescent microscope (Nikon Corp.). The results indicated that, in the probe array of this example, [0117]
(a) Each spot was almost round and had a diameter in a range of about 70-100 μm; [0118]
(b) There were spaces of about 100 μm, which was almost the same as the diameter of each spot, between adjacent spots so that each spot was clearly independent; [0119]
(c) The columns and rows of the spots were arranged in lines. [0120]
These facts are very effective in automatic detection, etc. of hybridized spots on a probe array. [0121]

EXAMPLE 2

Manufacturing of Nucleic Acid Probe Array Using Bubble Jet Printer and Detection of Target Nucleic Acid Using the Probe Array [0122]
(1) A glass plate for a probe array was prepared in the same manner as in (1) and (2) of Example 1. [0123]
(2) Synthesis of Probe DNA [0124]

Single-stranded nucleic acids of SEQ ID Nos. 1-4 were synthesized using an automatic DNA synthesizer. The ss nucleic acids of SEQ ID Nos. 2-4 were as follows: from the ss nucleic acid of SEQ ID No. 1 used in Example 1, one base differs in SEQ ID No. 2, 3 bases in SEQ ID No. 3, and 6 bases in SEQ ID No. 4. A thiol (—SH) group was introduced at each terminus of the ssDNAs of SEQ ID Nos. 1-4 using Thiol-Modifier (Glen Research Co., Ltd.) during synthesis on the automatic DNA synthesizer. Following ordinary deprotection, DNA was then recovered, purified with high performance liquid chromatography, and used in the following experiments. The sequences of SEQ ID Nos. 2-4 are shown below:


^5′HS-(CH₂)₆-O-PO₂-O-ACTGGCCGTTGTTTTACA^3′	SEQ ID
	No. 2:

^5′HS-(CH₂)₆-O-PO₂-O-ACTGGCCGCTTTTTTACA^3′	SEQ ID
	No. 3:

^5′HS-(CH₂)₆-O-PO₂-O-ACTGGCATCTTGTTTACA^3′	SEQ ID
	No. 4:

(3) DNA Probe Ejection and Binding to Substrate Using BJ Printer [0126]
The ssDNAs of SEQ ID Nos. 1-4 above were used to prepare 4 ejection liquids by the method similar to that described in (4) of Example 1. The respective liquids were filled in 4 ink tanks of a bubble jet printer used in Example 1 and the respective ink tanks were mounted on the bubble jet heads. The glass plate prepared in (1) was mounted on the printer, and the 4 nucleic acid probes were spotted in respective 4 areas of 3×3 mm on the glass plate. The spotting pattern in each area was the same as that in Example 1. After completion of spotting, the glass plate was left in a humidified chamber for 30 minutes to react the maleimido group and the thiol group. [0127]
(4) Blocking Reaction [0128]
After completion of the reaction between the maleimido group and the thiol group, the glass plate was washed with a 1 M NaCl/50 mM phosphate buffer solution (pH 7.0) to rinse completely away the solution containing DNA on the surface of the glass plate. [0129]
Then, the glass plate was immersed in a 2% bovine serum albumin aqueous solution and left for 2 hours to proceed a blocking reaction. [0130]
(5) Hybridization Reaction [0131]
A ssDNA with a base sequence complementary to DNA of SEQ ID No. 1 was synthesized using an automatic DNA synthesizer, and rhodamine was bound to its 5′-terminus to obtain a labeled ssDNA. This labeled ssDNA was dissolved in an 1 M NaCl/50 mM phosphate buffer solution (pH 7.0) to a final concentration of 1 μM. The probe array obtained in (4) was subjected to a hybridization reaction for 3 hours. Then, the probe array was washed with 1 M NaCl/50 mM phosphate buffer solution (pH 7.0) to wash away the ssDNA which had not been hybridized with the probe nucleic acid. Then, the respective spots of the probe array were observed using a fluorescent microscope (Nikon Corp.) and the amounts of fluorescence were quantified using the image analyzer (Product name: ARGUS; Hamamatsu Photonics Co., Ltd.). [0132]
(6) Results [0133]
The fluorescence intensity of the spots of the DNA probe of SEQ ID No. 1 completely matched with the labeled ssDNA was 4,600, while the fluorescence intensity was 2,800 for the spots of the DNA probe of SEQ ID No. 2 containing one mismatched base. For the spots of the DNA probe of SEQ ID No. 3 having 3 mismatched bases, the fluorescence intensity was 2,100, which was less than half that for the completely matched probe. No fluorescence was observed for DNA of SEQ ID No. 4 containing 6 mismatched bases. The above result indicates that a completely complementary ssDNA was specifically detected on the DNA array substrate. [0134]

EXAMPLE 3

Concentration of DNA Probe Solution and Bubble Jet Ejection Properties [0135]
(1) Synthesis of DNA Probe [0136]
A ssDNA of SEQ ID No. 5 shown below was synthesized using an automatic DNA synthesizer and dissolved in a TE solution (10 mM Tris-HCl (pH 8)/1 mM EDTA aqueous solution) to concentrations of about 0.2 mg/ml, 2 mg/ml, and 1.5 mg/ml to prepare DNA probe solutions of 3 different concentrations (accurate concentrations were calculated from absorbance). [0137]
SEQ ID No. 5: [0138]
[0139] ^5′GCCTGATCAGGC^3′
(2) Ejection by BJ Printer [0140]
An aqueous solution containing glycerin at 7.5 wt %, urea at 7.5 wt %, thiodiglycol at 7.5 wt %, and acetylene alcohol (Product name: Acetylenol EH; Kawaken Fine Chemical Co., Ltd.) having the above general formula (I) at 1 wt % was prepared, added to the 0.2 mg/ml probe solution prepared in (1), and adjusted a final concentration to about 0.02 mg/ml (3 μM). This solution was filled in an ink tank of a bubble jet printer used in Example 1 and the ink tank was mounted on a bubble jet head used in Example 1. [0141]
An aluminum plate of A4 size was mounted on the printer and the liquid was spotted to an area of 3×5 square inch of the aluminum plate. The condition of spotting was set so as to perform spotting in a density of 360×720 dpi in the above area. A commercial ink for BJ620 was first printed on the aluminum plate as a control. This operation was performed on a total of 4 aluminum plates. [0142]
The nucleic acid probe spotted on the respective aluminum plates was recovered using the TE solution and purified by a gel filtration method. The amounts of the recovered nucleic acid probe purified were measured by absorbance. The recovery of the nucleic acid probe theoretically obtained is as follows. That is, a volume of a droplet ejected from the head of the printer used in this example was 24 picoliters. Then, since there were 4 aluminum plates on which the solution was spotted in an area of 3×5 square inch at a density of 360×720 dpi, the following equation was obtained:[0143]
24 (picoliters)×(720×360)×(3×5)×4 plates=373 μl
Absorbance at 260 nm of the probe nucleic acid for this volume and absorbance at 260 nm of recovered nucleic acid probe are shown in FIG. 3. [0144]
(3) The operation identical to that described in (2) was performed on the probe solutions at concentrations of 2 mg/ml and 15 mg/ml. The final concentrations of the nucleic acid probe of the respective ejection liquids were 30 μM (0.2 mg/ml) and 225 μM (1.5 mg/ml). Absorbance of the probe nucleic acid recovered from the respective solutions and absorbance of the probe nucleic acid in amounts theoretically obtained are shown in FIG. 3. [0145]
(4) Results [0146]
As shown in FIG. 3, the amounts of a nucleic acid probe actually ejected were close to the values theoretically anticipated. From this, in ejection of a nucleic acid probe using the bubble jet method, no quantitative loss of the nucleic acid probe due to burning and sticking of the nucleic acid probe to the heater of the bubble jet head was observed. No troubles in the head, such as no ejection, occurred during the step of spotting on the aluminum plate using liquids of various concentrations. A macroscopic comparison with the spots of the ink for a bubble jet printer spotted on the aluminum plate as a control and the spots of the nucleic acid probe showed that the spotting status for the spots formed using the liquids at concentrations of 3 μM and 30 μM was similar to that for the ink spot. The spots formed using the liquid at a concentration of 225 μM exhibited some disorders as compared with the ink spot. [0147]

EXAMPLE 4

Influence of Bubble Jet Process on Nucleic Acid Probe [0148]
(1) Synthesis of Nucleic Acid Probe [0149]
A nucleic acid probe comprised of 10 mer adenylic acids (abbreviated as “A” hereinafter) (synthetic substance), oligoA (40-60 mer; Pharmacia Co., Ltd.), and poly(dA) (300-400 mer; Pharmacia Co., Ltd.) were respectively diluted with a TE solution to prepare solutions of the nucleic acid probes of different base lengths at a final concentration of 1 mg/ml. The base sequence of the 10-mer probe (SEQ ID No. 6) is shown below: [0150]
1 14 1 18 DNA Artificial Sequence modified_base 1 Thiol group bound at the 5′-terminus 1 acattttgct gccggtca 18 2 18 DNA Artificial Sequence modified_base 1 Thiol group bound at the 5′-terminus 2 acattttgtt gccggtca 18 3 18 DNA Artificial Sequence Synthetic 3 actggccgct tttttaca 18 4 18 DNA Artificial Sequence Synthetic 4 actggcatct tgtttaca 18 5 12 DNA Artificial Sequence Synthetic 5 gcctgatcag gc 12 6 10 DNA Artificial Sequence Synthetic 6 aaaaaaaaaa 10 7 18 DNA Artificial Sequence modified_base 1 Cysteine residue bound at the N′-terminus 7 actggccgtc gttttaca 18 8 18 DNA Artificial Sequence modified_base 1 Cysteine residue bound at the N′-terminus 8 actggccgtt gttttaca 18 9 18 DNA Artificial Sequence modified_base 1 Amino group bound at the 5′-terminus 9 tgaccggcag caaaatgt 18 10 18 DNA Artificial Sequence modified_base 1 Amino group bound at the 5′-terminus 10 tgaccggcac caaaatgt 18 11 18 DNA Artificial Sequence modified_base 1 Amino group bound at the 5′-terminus 11 tgacccgcac caatatgt 18 12 18 DNA Artificial Sequence modified_base 1 Thiol group bound at the 5′-terminus 12 tgacccgcag caaaatgt 18 13 18 DNA Artificial Sequence modified_base 1 Thiol group bound at the 5′-terminus 13 tgaccggcac caaaatgt 18 14 18 DNA Artificial Sequence modified_base 1 Thiol group bound at the 5′-terminus 14 tgacccgcac caatatgt 18

Claims

What is claimed is:

1. A method of spotting a probe which can bind specifically to a target to a solid support comprising the steps of:

supplying a liquid containing a probe on a surface of a solid support by an ink jet method and attaching the liquid, and

forming a spot of probe on the surface of the solid support.

2. The method of spotting according to claim 1 wherein the probe is a single-stranded nucleic acid probe.

3. The method of spotting according to claim 2 wherein the single-stranded nucleic acid probe includes a single-stranded DNA probe.

4. The method of spotting according to claim 2 wherein the single-stranded nucleic acid probe includes an RNA probe.

5. The method of spotting according to claim 2 wherein the single-stranded nucleic acid probe includes a single-stranded PNA probe.

6. The method of spotting according to claim 2 wherein the surface of the solid support has a first functional group and the single-stranded nucleic acid probe has a second functional group, and the first and second functional groups react each other by contact.

7. The method of spotting according to claim 6 wherein the first functional group on the surface of the solid support is a maleimido group and the second functional group of the single-stranded nucleic acid probe is a thiol (SH) group.

8. The method of spotting according to claim 7 wherein the solid support is a glass plate and the maleimido group is introduced by introducing an amino group on the surface of the glass plate and then reacting the amino group with N-(6-maleimidocaproyloxy) succinimide.

9. The method of spotting according to claim 7 wherein the solid support is a glass plate and the maleimido group is introduced by introducing an amino group on the surface of the glass plate and then reacting the amino group with succinimidyl-4-(maleimido phenyl) butyrate.

10. The method of spotting according to claim 7 wherein the maleimido group is reacted with the thiol group for at least 30 minutes.

11. The method of spotting according to claim 10 wherein the single-stranded nucleic acid comprises a single-stranded PNA probe, at the terminus of which the thiol group exists, and the maleimido group is reacted with the thiol group for at least 2 hours.

12. The method of spotting according to claim 11 wherein the thiol group at the terminus of the single-stranded PNA probe is introduced by binding cysteine group to the N-terminus of the single-stranded PNA probe.

13. The method of spotting according to claim 6 wherein the first functional group on the surface of the solid support is an epoxy group and the second functional group of the single-stranded nucleic acid probe is an amino group.

14. The method of spotting according to claim 13 wherein the solid support is a glass plate and the epoxy group is introduced by applying a silane compound having an epoxy group in the molecule thereof on the surface of the glass plate and reacting the compound with the glass plate.

15. The method of spotting according to claim 13 wherein the epoxy group is introduced by applying polyglycidyl methacrylate having an epoxy group on the solid support.

16. The method of spotting according to claim 1 wherein the liquid contains urea at 5-10 wt %, glycerin at 5-10 wt %, thiodiglycol at 5-10 wt %, and an acetylene alcohol at 1 wt % of the liquid.

17. The method of spotting according to claim 16 wherein the acetylene alcohol has a structure represented by the following general formula (I):

(wherein, R₁, R₂, R₃, and R₄represent an alkyl group, each m and n represent an integral, and m=0 and n=0 or 1≦m+n≦30, and when m+n=1, m or n is 0.)

18. The method of spotting according to claim 2 wherein a concentration of the single-stranded nucleic acid probe in the liquid is 0.05-500 μM.

19. The method of spotting according to claim 18 wherein a concentration of the single-stranded nucleic acid probe in the liquid is 2-50 μM.

20. The method of spotting according to claim 2 wherein a length of the single-stranded nucleic acid probe is 2-5,000 bases.

21. The method of spotting according to claim 20 wherein a length of the single-stranded nucleic acid probe is 2-60 bases.

22. The method of spotting according to claim 1 wherein the ink jet method is a bubble jet method.

23. The method of spotting according to claim 1 wherein the probe is an oligopeptide or a polypeptide with a specific amino acid sequence.

24. The method of spotting according to claim 1 wherein the probe is a protein.

25. The method of spotting according to claim 24 wherein the protein is an antibody.

26. The method of spotting according to claim 24 wherein the protein is an enzyme.

27. The method of spotting according to claim 1 wherein the probe is an enzyme.

28. The method of spotting according to claim 1 wherein the liquid is supplied so as to form independent spots in a density of 10,000 spots per square inch on the solid support.

29. The method of spotting according to claim 1 wherein the solid support has a flat surface and homogenous surface properties.

30. The method of spotting according to claim 29 wherein the liquid is supplied on the surface of the solid support so as to obtain a distance between the adjacent spots not smaller than the maximum width of the spot.

31. The method of spotting according to claim 30 wherein blocking is performed on the surface of the solid support to prevent a sample from attaching to the surface other than spots of the surface of the solid support.

32. The method of spotting according to claim 31 wherein the blocking is achieved by using bovine serum albumin.

33. The method of spotting according to claim 1 wherein the solid support is partitioned by a matrix arranged in a pattern on the surface, a plurality of wells whose bottom is the surface of the solid support exposed in the pattern are provided, and the liquid is supplied to the respective wells.

34. The method of spotting according to claim 33 wherein the solid support is optically transparent and the matrix is opaque.

35. The method of spotting according to claim 33 wherein the matrix comprises a resin.

36. The method of spotting according to claim 33 wherein the surface of the matrix is hydrophobic.

37. The method of spotting according to claim 33 wherein the bottom of the wells is hydrophilic.

38. The method of spotting according to claim 33 wherein the matrix has a thickness of 1-20 μm.

39. The method of spotting according to claim 33 wherein the wells have a maximum width of 200 μm.

40. The method of spotting according to claim 33 wherein the matrix has a width 1/2-2 times the maximum width of the wells.

41. A probe array comprising a plurality of spots of a probe, the spots being provided independently at a plurality of sites of a surface of a solid support in a density of 10,000 spots per square inch or higher.

42. The probe array according to claim 41 wherein the solid support has a flat surface and homogenous surface properties.

43. The probe array according to claim 42 wherein the probe is a single-stranded nucleic acid probe.

44. The probe array according to claim 43 wherein the single-stranded nucleic acid probe includes a single-stranded DNA probe.

45. The probe array according to claim 43 wherein the single-stranded nucleic acid includes a single-stranded RNA probe.

46. The probe array according to claim 43 wherein the single-stranded nucleic acid includes a single-strand ed PNA probe.

47. The probe array according to claim 43 wherein the single-stranded nucleic acid is covalently bound to the surface of the solid support by a reaction between a first functional group on the surface of the solid surface and a second functional group of the single-stranded nucleic acid probe.

48. The probe array according to claim 47 wherein the first functional group on the surface of the solid support is a maleimido group and the second functional group of the single-stranded nucleic acid probe is a thiol (SH) group.

49. The probe array according to claim 48 wherein the single-stranded nucleic acid probe is a single-stranded PNA probe and contains a cysteine residue on an N-terminus side.

50. The probe array according to claim 47 wherein the first functional group on the surface of the solid support is an epoxy group and the second functional group of the single-stranded nucleic acid probe is an amino group.

51. The probe array according to claim 42 wherein the spots are formed by supplying a liquid containing the probe on the solid support.

52. The probe array according to claim 42 wherein the probe is an oligopeptide or a polypeptide with a specific amino acid sequence.

53. The probe array according to claim 42 wherein the probe is a protein.

54. The probe array according to claim 53 wherein the protein is an antibody.

55. The probe array according to claim 53 wherein the protein is an enzyme.

56. The probe array according to claim 42 wherein the probe is an antigen.

57. The probe array according to claim 42 wherein a distance between the adjacent spots is not smaller than a maximum width of the spot.

58. The probe array according to claim 41 wherein the solid support is partitioned by a matrix arranged in a pattern on the surface, a plurality of wells whose bottom is the surface of the solid support exposed in a pattern are provided, and the liquid is supplied to the respective wells.

59. The probe array according to claim 58 wherein the probe is a single-stranded nucleic acid probe.

60. The probe array according to claim 59 wherein the single-stranded nucleic acid probe includes a single-stranded DNA probe.

61. The probe array according to claim 59 wherein the single-stranded nucleic acid includes a RNA probe.

62. The probe array according to claim 59 wherein the single-stranded nucleic acid includes a single-stranded PNA probe.

63. The probe array according to claim 62 wherein the single-stranded nucleic acid is covalently bound to the surface of the solid support by a reaction between the first functional group of the surface of the solid surface and the second functional group on the single-stranded nucleic acid probe.

64. The probe array according to claim 63 wherein the first functional group on the surface of the solid support is a maleimido group and the second functional group of the single-stranded nucleic acid probe is a thiol (SH) group.

65. The probe array according to claim 64 wherein the single-stranded nucleic acid probe is a single-stranded PNA probe and contains a cysteine residue on an N-terminal side.

66. The probe array according to claim 63 wherein the first functional group on the surface of the solid support is an epoxy group and the second functional group of the single-stranded nucleic acid probe is an amino group.

67. The probe array according to claim 58 wherein the spots are formed by supplying a liquid containing a probe on the solid support.

68. The probe array according to claim 58 wherein the probe is an oligopeptide or a polypeptide with a specific amino acid sequence.

69. The probe array according to claim 58 wherein the probe is a protein.

70. The probe array according to claim 69 wherein the protein is an antibody.

71. The probe array according to claim 69 wherein the protein is an enzyme.

72. The probe array according to claim 58 wherein the probe is an antigen.

73. The probe array according to claim 58 wherein the matrix is opaque.

74. The probe array according to claim 73 wherein the solid support is optically transparent.

75. The probe array according to claim 58 wherein the matrix comprises a resin.

76. The probe array according to claim 58 wherein the probe is attached only to the wells.

77. The probe array according to claim 58 wherein the matrix has a thickness of 1-20 μm.

78. The probe array according to claim 58 wherein the wells have a maximum width of 200 μm.

79. The probe array according to claim 58 wherein a distance between the wells is 1/2-2 times the maximum width of the wells.

80. The probe array according to claim 41 wherein the probe array comprises at least 2 spots each of which comprises a different kind of probe.

81. A method of manufacturing a probe array having a plurality of spots arranged independently in a plurality of sites on a surface of a solid support, the spots containing a probe which can bind specifically to a target substance comprising a step of supplying a liquid containing the probe and attaching the liquid to a predetermined site on the surface of the solid support by means of an ink jet method to form the spots.

82. The method of manufacturing according to claim 81 wherein the probe is a single-stranded nucleic acid probe.

83. The method of manufacturing according to claim 82 wherein the single-stranded nucleic acid probe is a single-stranded DNA probe.

84. The method of manufacturing according to claim 82 wherein the single-stranded nucleic acid probe is an RNA probe.

85. The method of manufacturing according to claim 82 wherein the single-stranded nucleic acid probe is a single-stranded PNA probe.

86. The method of manufacturing according to claim 82 wherein the surface of the solid surface has a first functional group and the single-stranded nucleic acid probe has a second functional group, and the first and the second functional groups react each other by contact.

87. The method of manufacturing according to claim 86 wherein the first functional group on the surface of the solid support is a maleimido group and the second functional group of the single-stranded nucleic acid probe is a thiol (SH) group.

88. The method of manufacturing according to claim 87 wherein the solid support is a glass plate and a maleimido group is introduced by introducing an amino group on the surface of the glass plate and then reacting the amino group with N-(6-maleimidocaproyloxy) succinimide.

89. The method of manufacturing according to claim 87 wherein the solid support is a glass plate and the maleimido group is introduced by introducing an amino group on the surface of the glass plate and then reacting the amino group with succinimidyl-4-(maleimido phenyl) butyrate.

90. The method of manufacturing according to claim 87 wherein the maleimido group is reacted with the thiol group for at least 30 minutes.

91. The method of manufacturing according to claim 90 wherein the single-stranded nucleic acid is a single-stranded PNA probe, at the terminus of which the thiol group exists, the maleimido group is reacted with the thiol group for at least 2 hours.

92. The method of manufacturing according to claim 91 wherein the thiol group at the terminus of the single-stranded PNA probe is introduced by binding cysteine group to an N-terminal of the single-stranded PNA probe.

93. The method of manufacturing according to claim 86 wherein the first functional group on the surface of the solid support is an epoxy group and the second functional group of the single-stranded nucleic acid probe has is an amino group.

94. The method of manufacturing according to claim 93 wherein the solid support is a glass plate and the epoxy group is introduced by applying a silane compound having an epoxy group in the molecule thereof on the surface of the glass plate and reacting the compound with the glass plate.

95. The method of manufacturing according to claim 93 wherein the epoxy group is introduced by applying polyglycidyl methacrylate having an epoxy group on the solid support.

96. The method of manufacturing according to claim 93 wherein the liquid contains urea at 5-10 wt %, glycerin at 5-10 wt %, thiodiglycol at 5-10 wt %, and an acetylene alcohol at 1 wt % of the liquid.

97. The method of manufacturing according to claim 96 wherein the acetylene alcohol has a structure represented by the following general formula (I):

(wherein, R₁, R₂, R₃, and R₄represent an alkyl group, each m and n represent an integral, and m=0 and n=0, or 1≦m+n≦30, and when m+n=1, m or n is 0.)

98. The method of manufacturing according to claim 82 wherein a concentration of the single-stranded nucleic acid probe in the liquid is 0.05-500 μM.

99. The method of manufacturing according to claim 98 wherein the concentration of the single-stranded nucleic acid probe in the liquid is 2-50 μM.

100. The method of manufacturing according to claim 82 wherein a length of the single-stranded nucleic acid probe is 2-5,000 bases.

101. The method of manufacturing according to claim 100 wherein the length of the single-stranded nucleic acid probe is 2-60 bases.

102. The method of manufacturing according to claim 81 wherein the ink jet method is a bubble jet method.

103. The method of manufacturing according to claim 81 wherein the liquid is supplied so as to form independent spots in a density of 10,000 spots per square inch on the solid support of higher.

104. The method of manufacturing according to claim 81 wherein the probe is an oligopeptide or a polypeptide with a specific amino acid sequence.

105. The method of manufacturing according to claim 81 wherein the probe is a protein.

106. The method of manufacturing according to claim 105 wherein the protein is an antibody.

107. The method of manufacturing according to claim 105 wherein the protein is an enzyme.

108. The method of manufacturing according to claim 81 wherein the probe is an antigen.

109. The method of manufacturing according to claim 81 wherein the solid support has a flat surface and homogenous surface properties.

110. The method of manufacturing according to claim 109 wherein blocking is performed, following spotting of the probe to the solid support, to prevent a sample from attaching to the surface other than the spots.

111. The method of manufacturing according to claim 110 wherein the blocking comprises a step of immersing the solid support to which the spots have been formed in an aqueous solution of bovine serum albumin.

112. The method of manufacturing according to claim 111 wherein a concentration of the aqueous solution of bovine serum albumin is 0.1-5%.

113. The method of manufacturing according to claim 111 wherein the solid support is immersed in an aqueous solution of bovine serum albumin for at least 2 hours.

114. The method of manufacturing according to claim 109 wherein the liquid is supplied on the surface of the solid support so as to obtain a distance between the adjacent spots not smaller than a maximum width of the spot.

115. The method of manufacturing according to claim 81 wherein the solid support is partitioned by a matrix arranged in a pattern on the surface, a plurality of wells whose bottom is the surface of the solid support exposed in a pattern are provided, and the liquid is supplied to the respective wells.

116. The method of manufacturing according to claim 115 wherein the solid support is optically transparent and the matrix is opaque.

117. The method of manufacturing according to claim 115 wherein the matrix comprises a resin.

118. The method of manufacturing according to claim 115 wherein the surface of the matrix is hydrophobic.

119. The method of manufacturing according to claim 115 wherein the bottom of the wells is hydrophilic.

120. The method of manufacturing according to claim 115 wherein the wells have a maximum width of 200 μm.

121. The method of manufacturing according to claim 115 wherein the matrix has a width 1/2-2 times the maximum width of the wells.

122. The method of manufacturing according to claim 115 wherein a thickness of the matrix is 1-20 μm.

123. The method of manufacturing according to claim 115 wherein the matrix pattern is formed by photolithography.

124. The method of manufacturing according to claim 123 wherein the photolithography comprises a step of forming a resin layer on the surface of the solid support, forming a photoresist layer on the resin layer, exposing the photoresist layer to light in a pattern corresponding to the matrix pattern, and developing to form the pattern of the photoresist on the resin layer; and a step of patterning the resin layer using the pattern of the photoresist as a mask and then removing the pattern of the photoresists.

125. The method of manufacturing according to claim 123 wherein the photolithography comprises the steps of forming a photosensitive resin layer on the surface of the solid support, exposing the photosensitive resin layer to light in a pattern corresponding to the matrix pattern, and developing.

126. The method of manufacturing according to claim 125 wherein the photosensitive resin layer is selected from the group consisted of a UV resist, a DEEP-UV resist, or an ultraviolet cure resin.

127. The method of manufacturing according to claim 126 wherein the UV resist is selected from the group consisted of a cyclized polyisoprene-aromatic bisazide resist, a phenol resin-aromatic azide compound resist, or a novolak resin-diazonaphtoquinone resist.

128. The method of manufacturing according to claim 126 wherein the DEEP-UV resin is a radiolysable polymer resist or a dissolution suppressant resist.

129. The method of manufacturing according to claim 128 wherein the radiation decomposition polymer resist is at least one selected from a group consisting of polymethyl methacrylate, polymethylene sulfone, polyhexafluorobutyl methacrylate, polymethylisopropenyl ketone, and poly-1-trimethylsilyl propyne bromide.

130. The method of manufacturing according to claim 128 wherein the dissolution suppressant resist is o-nitrobenzyl cholate ester.

131. The method of manufacturing according to claim 126 wherein the DEEP-UV resist is polyvinylphenol-3,3′-diazidediphenyl sulfone or polyglycidyl polymethacrylate.

132. The method of manufacturing according to claim 125 wherein water repellency of the matrix pattern formed by patterning of the photosensitive resin layer is further improved by postbaking of the matrix pattern.

133. The method of manufacturing according to claim 115 wherein a first functional group which can form a covalent bond with a second functional group of the probe is introduced on the surface of the solid support prior to formation of the wells.

134. The method of manufacturing according to claim 115 wherein a first functional group which can form a covalent bond with a second functional group of the probe is introduced on the surface of the solid support following formation of the wells.

135. The method of manufacturing according to claim 134 wherein a solution containing a compound for introducing the first functional group to the surface of the solid support is supplied to the wells.

136. The method of manufacturing according to claim 135 wherein the solution is supplied to the wells by means of the ink jet method.

137. The method of manufacturing according to claim 136 wherein the solution is a silane coupling agent containing a silane compound having an epoxy group or an amino group in its molecule.

138. The method of manufacturing according to claim 136 wherein the solution contains a compound which can react with an amino group on a glass substrate to introduce a maleimido group on the glass substrate.

139. The method of manufacturing according to claim 138 wherein the compound is N-maleimidocaproyloxy succinimide or succinimidyl-4-(maleimidophenyl) butyrate.

140. A method for detecting whether a target substance is contained in a sample, comprising the steps of:

providing a probe array comprising a plurality of spots each containing a probe which specifically binds to the target substance, the spots being arranged independently on a solid support;

contacting the sample with each of the spots; and

detecting presence or absence of a reacted product between the target substance and the probe, wherein the respective spots are formed by spotting a liquid containing the probe on the solid support by an ink jet method.

141. The method according to claim 140 wherein the target substance is a single-stranded nucleic acid having a first base sequence and the probe is a single-stranded nucleic acid probe having a second base sequence complementary to the first base sequence.

142. The method according to claim 141 wherein the single-stranded nucleic acid probe is a single-stranded DNA probe.

143. The method according to claim 141 wherein the single-stranded nucleic acid probe is an RNA probe.

144. The method according to claim 141 wherein the single-stranded nucleic acid probe is a single-stranded PNA probe.

145. The method according to claim 141 wherein the surface of the solid surface has a first functional group and the single-stranded nucleic acid probe has a second functional group, respectively, and the functional groups react each other by contact.

146. The method according to claim 145 wherein the first functional group on the surface of the solid support is a maleimido group and the second functional group of the single-stranded nucleic acid probe is a thiol (SH) group.

147. The method according to claim 146 wherein the solid support is a glass plate and the maleimido group is introduced by introducing an amino group on the surface of the glass plate and then reacting the amino group with N-(6-maleimidocaproyloxy) succinimide.

148. The method according to claim 146 wherein the solid support is a glass plate and the maleimido group is introduced by introducing an amino group on the surface of the glass plate and then reacting the amino group with succinimidyl-4-(maleimidophenyl) butyrate.

149. The method according to claim 146 wherein the maleimido group is reacted with the thiol group for at least 30 minutes.

150. The method according to claim 149 wherein the single-stranded nucleic acid comprises a single-stranded PNA probe having a thiol group on the terminus and the maleimido group is reacted with the thiol group for at least 2 hours.

151. The method according to claim 146 wherein the thiol group at a terminus of the single-stranded PNA probe is introduced by binding cysteine group to an N-terminus of the single-stranded PNA probe.

152. The method according to claim 145 wherein the first functional group on the surface of the solid support is an epoxy group and the second functional group of the single-stranded nucleic acid probe is an amino group.

153. The method according to claim 152 wherein the solid support is a glass plate and the epoxy group is introduced by applying a silane compound having an epoxy group in the molecule thereof on the surface of the glass plate and reacting the compound with the glass plate.

154. The method according to claim 152 wherein the epoxy group is introduced by applying polyglycidyl methacrylate having an epoxy group on the solid support.

155. The method according to claim 141 wherein the liquid contains urea at 5-10 wt %, glycerin at 5-10 wt %, thiodiglycol at 5-10 wt %, and an acetylene alcohol at 1 wt % of the liquid.

156. The method according to claim 155 wherein the acetylene alcohol has a structure represented by the following general formula (I):

157. The method according to claim 155 wherein a concentration of the single-stranded nucleic acid probe in the liquid is 0.05-500 μM.

158. The method according to claim 157 wherein a concentration of the single-stranded nucleic acid probe in the liquid is 2-50 μM.

159. The method according to claim 155 wherein a length of the single-stranded nucleic acid probe is 2-5,000 bases.

160. The method according to claim 159 wherein a length of the single-stranded nucleic acid probe is 2-60 bases.

161. The method according to claim 141 wherein the ink jet method is a bubble jet method.

162. The method according to claim 140 wherein the probe is an oligopeptide or a polypeptide with a specific amino acid sequence.

163. The method according to claim 140 wherein the probe is a protein.

164. The method according to claim 163 wherein the protein is an antibody.

165. The method according to claim 163 wherein the protein is an enzyme.

166. The method according to claim 140 wherein the probe is an antigen.

167. The method according to claim 140 wherein the liquid is supplied so as to form independent spots in a density of 10,000 spots per square inch on the solid support.

168. The method according to claim 140 wherein the solid support has a flat surface and homogenous surface properties.

169. The method according to claim 168 wherein the liquid is supplied on the surface of the solid support so as to obtain a distance between the adjacent spots not smaller than the maximum width of the spots.

170. The method according to claim 168 wherein blocking is performed on the surface of the solid support to prevent the sample from attaching to the surface other than the spots of the surface of the solid support.

171. The method according to claim 170 wherein blocking is achieved by using bovine serum albumin.

172. The method according to claim 140 wherein the solid support is partitioned by a matrix arranged in a pattern on the surface, a plurality of wells whose bottom is the surface of the solid support exposed in the pattern are provided, and the liquid is supplied to the respective wells.

173. The method according to claim 172 wherein the solid support is optically transparent and the matrix is opaque.

174. The method according to claim 172 wherein the matrix comprises a resin.

175. The method according to claim 172 wherein the surface of the matrix is hydrophobic.

176. The method according to claim 172 wherein the bottom of the wells is hydrophilic.

177. The method according to claim 172 wherein the matrix has a thickness of 1-20 μm.

178. The method according to claim 172 wherein the wells have a maximum width of 200 μm.

179. The method according to claim 172 wherein the matrix has a width 1/2-2 times the maximum width of the wells.

180. A method of identifying a structure of a target substance contained in a sample comprising the steps of:

preparing a probe array provided with spots of a probe, the probe being able to bind specifically to the target substance, on a surface of a solid support;

contacting the sample to the spots; and

detecting binding between the target substance and the probe.

181. The method of identification according to claim 180 wherein the target substance is a single-stranded nucleic acid, the structure to be identified is a base sequence of the single-stranded nucleic acid as the target substance,

the probe array is provided with a plurality of spots each of which contains single-stranded nucleic acids with different base sequences on a solid support, at least one of the spots contain a single-stranded nucleic acid with a base sequence complementary to that anticipated for the single-stranded nucleic acid as the target substance, and the plurality of spots are formed by attaching a liquid containing the respective single-stranded nucleic acids on the solid support by means of an ink jet method.

182. The method of identification according to claim 181 wherein the single-stranded nucleic acid probe is a single-stranded DNA probe.

183. The method of identification according to claim 181 wherein the single-stranded nucleic acid probe is an RNA probe.

184. The method of identification according to claim 181 wherein the single-stranded nucleic acid probe is a single-stranded PNA probe.

185. The method of identification according to claim 181 wherein the surface of the solid surface and the single-stranded nucleic acid probe have a first and a second functional groups, respectively, and the functional groups react each other by contact.

186. The method of identification according to claim 181 wherein the first functional group on the surface of the solid support is a maleimido group and the second functional group of the single-stranded nucleic acid probe is a thiol (SH) group.

187. The method of identification according to claim 186 wherein the solid support is a glass plate and the maleimido group is introduced by introducing an amino group on the surface of the glass plate and then reacting the amino group with N-(6-maleimidocaproyloxy) succinimide.

188. The method of identification according to claim 186 wherein the solid support is a glass plate and the maleimido group is introduced by introducing an amino group on the surface of the glass plate and then reacting the amino group with succinimidyl-4-(maleimido phenyl) butyrate.

189. The method of identification according to claim 186 wherein the maleimido group is reacted with the thiol group for at least 30 minutes.

190. The method of identification according to claim 189 wherein the single-stranded nucleic acid is a single-stranded PNA probe having a thiol group on the terminus thereof and the maleimido group is reacted with the thiol group for at least 2 hours.

191. The method of identification according to claim 186 wherein the thiol group at the terminus of the single-stranded PNA probe is introduced by binding cysteine group to an N-terminus of the single-stranded PNA probe.

192. The method of identification according to claim 185 wherein the first functional group on the surface of the solid support is an epoxy group and the second functional group of the single-stranded nucleic acid probe is an amino group.

193. The method of identification according to claim 192 wherein the solid support is a glass plate and the epoxy group is introduced by applying a silane compound having an epoxy group in the molecule on the surface of the glass plate and reacting the compound with the glass plate.

194. The method of identification according to claim 192 wherein the epoxy group is introduced by applying polyglycidyl methacrylate having an epoxy group on the solid support.

195. The method of identification according to claim 181 wherein the liquid contains urea at 5-10 wt %, glycerin at 5-10 wt %, thiodiglycol at 5-10 wt %, and acetylene alcohol at 1 wt % of the liquid.

196. The method of identification according to claim 195 wherein the acetylene alcohol has a structure represented by the following general formula (I):

197. The method of identification according to claim 195 wherein a concentration of the single-stranded nucleic acid probe in the liquid is 0.05-500 μM.

198. The method of identification according to claim 197 wherein the concentration of the single-stranded nucleic acid probe in the liquid is 2-50 μM.

199. The method of identification according to claim 197 wherein a length of the single-stranded nucleic acid probe is 2-5,000 bases.

200. The method of identification according to claim 199 wherein a length of the single-stranded nucleic acid probe is 2-60 bases.

201. The method of identification according to claim 181 wherein the ink jet method is a bubble jet method.

202. The method of identification according to claim 180 wherein the probe is an oligopeptide or a polypeptide with a specific amino acid sequence.

203. The method of identification according to claim 180 wherein the probe is a protein.

204. The method of identification according to claim 203 wherein the protein is an antibody.

205. The method of identification according to claim 203 wherein the protein is an enzyme.

206. The method of identification according to claim 180 wherein the probe is an antigen.

207. The method of identification according to claim 180 wherein the liquid is supplied so as to form independent spots in a density of 10,000 spots per square inch on the solid support.

208. The method of identification according to claim 180 wherein the solid support has a flat surface and homogenous surface properties.

209. The method of identification according to claim 208 wherein the liquid is supplied on the surface of the solid support so as to obtain a distance between the adjacent spots not smaller than the maximum width of the spots.

210. The method of identification according to claim 208 wherein blocking is performed on the surface of the solid support to prevent the sample from attaching to the surface other than spots of the surface of the solid support.

211. The method of identification according to claim 210 wherein blocking is achieved by using bovine serum albumin.

212. The method of identification according to claim 180 wherein the solid support is partitioned by a matrix arranged in a pattern on the surface, a plurality of wells whose bottom is the surface of the solid support exposed in the pattern are provided, and the liquid is supplied to the respective wells.

213. The method of identification according to claim 212 wherein the solid support is optically transparent and the matrix is opaque.

214. The method of identification according to claim 212 wherein the matrix comprises a resin.

215. The method of identification according to claim 212 wherein the surface of the matrix is hydrophobic.

216. The method of identification according to claim 212 wherein the bottom of the wells is hydrophilic.

217. The method of identification according to claim 212 wherein the matrix has a thickness of 1-20 μm.

218. The method of identification according to claim 212 wherein the wells have a maximum width of 200 μm.

219. The method of identification according to claim 212 wherein the matrix has a width 1/2-2 times a maximum width of the wells.