JP2002065299A - Simultaneous multi-item multiple sample inspection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a process for multi-item multiple sample inspection by preparing a matrix substrate to which biological specimens of different properties and different origins are bonded and spotting oligonucleotides having different sequences, proteins and medicines on the array, in order to inspect multiple samples simultaneously on the multi-items. SOLUTION: The first specimen, for example, a biological sample, is immobilized overall the surface of the matrix substrate within a preliminarily specified area, then another specifimen comprising different second and layer reagents is applied in this area in the form of individually independent spots to inspect the reactivity between dindividual samples whereby the multi-item inspection of the biological specimens can be simultaneously and efficiently carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention aims at simultaneously testing multiple specimens of a plurality of specimens, producing a matrix substrate on which biological samples having different properties and origins are combined, and further providing a different array on each matrix. By spotting oligonucleotides, proteins, and drugs on an array.

[0002]

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

[0003] A general method for manufacturing such a probe array is described, for example, in EP 373203.
As described in Japanese Unexamined Patent Application Publication No. (EP 0373203B1), a method of synthesizing a nucleic acid probe on a solid phase, a method of supplying a previously synthesized nucleic acid probe on a solid phase, and the like are known. As a prior art in which the former method is disclosed, for example, US Pat. No. 5,405,783 (US Pat. No. 5,405,783) is cited.

As the latter method, for example, US Pat. No. 5,601,980 (US Pat. No. 5,601,980)
And "Science", Vol. 270, 4
P. 67, (1995) discloses a method of arranging cDNAs in an array using micropipetting.

In these methods, 10,000 or more nucleic acid probes are arranged at a high density per square inch on a solid phase. Such a high-density DNA array has the advantage that a small number of samples can be tested simultaneously for many items, and the burden associated with collecting samples from a subject is small.

[0006]

As a method of producing the high-density DNA array on a substrate by a synthesis method, a method using a photolithography technique is disclosed in the aforementioned US Pat. No. 5,405,783. Performing this method requires sophisticated equipment, and not everyone can easily produce it. When the number of required test items is small and the number of samples is large, the degree of integration of DNA probes on the DNA array does not need to be very high. Rather, DN in which the desired DNA probe is immobilized more easily.
An A array may be required.

[0007] For example, 100
Inspection of items exceeding 00 is not always necessary.
As in mass screening, it may be important to examine a large number of specimens for a particular item. In such a case, a system is required that can rapidly test for the presence or absence of a disease in a larger number of samples than in a comparative example with a standard sample.

Furthermore, the amount of a specimen DNA is generally smaller than that of a synthesizable oligonucleotide. As in the usual hybridization reaction, DN
A reaction in which the A array substrate is immersed in the sample solution requires sufficient sample DNA to immerse the substrate. For that, D
The size of the NA array substrate is limited, and high density is required. Alternatively, in order to secure the volume of the solution, a method of diluting with a solvent to lower the concentration of the sample solution and lengthening the reaction time is adopted.

In addition, since the sample is separated or extracted from the tissue, the amount of the sample is limited, and the pretreatment used for the hybridization reaction (not only the extraction of nucleic acid, but also the treatment of single-strand and labeling) Is added), so that the obtained sample amount is further reduced. for that reason,
Recently, it has been used for inspection and research after an amplification process such as a PCR process. However, performing a PCR reaction has a disadvantage that only a specific gene can be examined since a primer is required. Furthermore, there are sequences that are easily amplified and those that are hardly amplified in the course of the PCR reaction, and the efficiency of the reaction is not uniform. Therefore, specific mRN in the extracted total mRNA
In order to check the content of A and determine a disease or the like, it is necessary to always prepare a reference sample.

[0010] The smaller the size, the smaller the required amount of the sample solution, but there is a physical limit in minimizing the substrate from the viewpoint of its handleability. In short, we need to increase the density,
It is possible to reduce the size of the substrate by reducing the number of probes, but in the course of the hybridization reaction and its detection, etc., a substrate that is too small is practical because it requires equipment for handling. I can't say.

[0011] Further, a method using a cDNA array (for example, the above-mentioned "Science", No. 270)
Volume, p. 467, (1995)) uses a DNA array in which various types of specimens are arranged, such as cDNA in the developmental process of a certain organism and cDNA in each phase in the culture process of a certain cell. Even in this method, in many cases, 10
More than 000 cDNAs are arranged as an array.
However, depending on the type of study, it is not always necessary to examine 10,000 species simultaneously. Before extracting such a large amount of cDNA, it is also necessary to conduct a simple study with a small number of samples.

Further, in this method, a labeled DNA having a specific function and a known sequence is reacted with a probe solution as a usual method. However, when a plurality of items are examined, the number of items is reduced. Only requires the number of arrays. In order to avoid this and to consider a plurality of items on the same array, the number of types of fluorescent reagents used for labeling is required. Since these fluorescent dyes must be detected separately at the time of detection, their wavelengths and the like need to be different, and a filter for detection corresponding to each is also required, which makes it difficult to select a dye. .

The need for such a multi-item, multi-sample simultaneous test is not limited to a hybridization reaction between genes.

For example, the interaction between a gene and a protein trade (D
There are some needs for interaction with genes, such as screening for chemical substances that bind to genes (NA binding protein) and genes. The former is used to elucidate the control mechanism of a gene by a protein, but at present, a method is used in which a gene and a protein are combined and then analyzed by gel electrophoresis. According to this method, the number of samples that can be analyzed at one time is limited.

In the field of drug discovery, it is important to examine the interaction between a gene and a drug. Obtaining a chemically synthesized product is quite time-consuming, and it is considered that if the amount used for screening can be reduced, the efficiency can be significantly improved.

[0016]

SUMMARY OF THE INVENTION An object of the present invention is to provide a multi-item test for a plurality of specimens simultaneously, for example, to prepare a matrix substrate on which biological samples having different properties and origins are combined,
It is still another object of the present invention to provide a method for simultaneously testing multiple specimens by spotting oligonucleotides, proteins, and drugs having different sequences on each matrix on an array.

Another object of the present invention is to provide a method for simultaneously examining a large number of specimens for a plurality of items for the purpose of interaction between a chemical substance, particularly a drug and cDNA, and binding between a protein and cDNA. is there.

[0018]

According to the present invention, there is provided a method for simultaneously testing the reactivity of a first sample with a plurality of samples, the method comprising: Are defined as areas smaller than the defined area in each of the second to second + nth (n ≧ 1) samples having different properties in a defined area on the substrate which is previously bonded over the entire surface. Independently arranged, the first
And the second to second + n (n ≧ 1) samples are tested for reactivity.

In the biological sample matrix according to the present invention, which is useful for such an inspection method, a plurality of types of biological samples having different origins are present on each matrix partitioned on a substrate. It is characterized by the following.

According to the present invention, it is possible to provide a substrate on which biological samples (for example, nucleic acids and proteins) having different properties and origins are previously bound in a matrix.

Further, as one form of the substrate, there is provided a substrate in which biological samples having different properties and origins are bonded on a matrix previously separated by a pattern with a hydrophobic compound, and a method for producing the same. it can.

On the substrate on which biological samples having different properties and origins are arranged in a matrix, another sample such as a DNA probe such as an oligonucleotide, a cDNA, a protein, or a chemical substance is spotted in an array. By performing the reaction in the same manner, the presence or absence of the binding of another sample to a specific biological sample,
In addition, it is possible to provide a method for quickly checking.

More specifically, when the samples arranged in a matrix are cDNAs, a hybridization reaction is performed by spotting various types of oligonucleotide probes on the substrate in an array. This provides a method for rapidly examining the complementarity of various nucleic acid probes to a specific cDNA. Also,
When the sample to be spotted is a plurality of types of proteins, it becomes possible to screen for proteins that bind to a specific cDNA. Furthermore, in the case of a drug, screening for a drug that can interact with a specific cDNA is performed.

In this method, since a plurality of types of samples are arranged on one substrate, the area occupied by one sample is extremely small. Therefore, a huge number of D
D with NA probes pre-bound in an array
There is an advantage that the amount of cDNA required is extremely small as compared with the case where a hybridization reaction is performed using an NA array. Further, there is no limitation on the size of the DNA array substrate, and there is no inconvenience in handling.

Further, by providing a method capable of testing even a small number of samples, a new testing area in which a sufficient sample amount has not been obtained so far and which could not be examined, such as directly examining mRNA obtained from a tissue, has been proposed. Open the way to

Further, according to the present invention, it is possible to provide a method capable of simultaneously examining chemical substances, proteins, and nucleic acids under the same reaction conditions on the same substrate.

When a biological sample arranged in a matrix is a specific protein, if the sample to be spotted is various nucleic acids, screening of a nucleic acid capable of binding to the specific protein is performed. This is to detect the interaction between the two, and if it is a drug, it is to screen for a drug that binds to a specific protein.

Which substance is arranged in the form of a matrix and which substance is arranged in the form of a spot differs depending on the purpose to be examined. Also, the availability of samples is an important factor. It is more effective to arrange a matrix which is easy to prepare in a large amount, but the sample arranged in a matrix needs to be fixed to the substrate by chemical bonding or adsorption. It is also important that the sample be able to withstand drying during the fixing process. Drugs that are only soluble in volatile solvents are unsuitable for spot sample delivery and should be delivered in a matrix.

[0029]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described below with reference to FIG. FIG. 1 shows a substrate surface on which 64 defined regions are formed. Each region (matrix) is 1 mm × 1 mm, and an interval x between the regions can be arbitrarily selected. For example, as a method for producing a biological sample-bound matrix substrate, a solution of a first sample (for example, a biological sample) is applied over the entire surface of a defined area on the substrate, printed as a “solid pattern” by an inkjet method, Alternatively, it is supplied by a method such as chemical synthesis on the substrate, and is bonded to the substrate in a matrix form by adsorption on the substrate or a chemical reaction between the functional group present in the biological sample and the functional group on the substrate. Methods are available. Note that the state in which the first sample is bonded to the entire surface of the defined area means that when the second and subsequent samples are supplied to the defined area, the sample is limited to the supply position in the area. Rather, it refers to a state in which the first sample is bonded over the entire surface to the extent that these reactions occur. For example, a state in which the first sample is fixed in a layered manner over the entire surface, or a state in which molecules and clumps constituting the first sample are dispersed at high density at minute intervals.

The defined area on the substrate may be provided on the substrate in advance as a well composed of sections partitioned in a pattern by walls of a hydrophobic compound.

Further, as a first sample, using a substrate on which a nucleic acid (cDNA) as a biological sample is immobilized, cDN
Multiple types of probes D that may be contained in A
When the NA is brought into contact with the cDNA on the substrate as the second and subsequent samples and the reaction product with the probe is detected on the solid phase to detect the presence or absence of the probe DNA sequence in the cDNA, various cDNAs are used. By supplying a plurality of types of probes in an array form as spots independent of each other in each matrix bound to the region defined by, the simultaneous assay with a plurality of types of probes becomes possible.

Further, on a nucleic acid (cDNA) matrix,
By bringing a plurality of types of chemical substances or proteins that may bind to cDNA into contact with the probe DNA on the substrate as independent spots, it is possible to simultaneously perform a multi-item inspection consisting of these reactions. By detecting the presence or absence of a bond between a protein and a probe on a solid phase as described above, a DNA-binding protein and a DNA-binding chemical can be simultaneously screened for multiple items.

The present invention is characterized in that probe DNAs, proteins, and chemical substances are supplied in the form of microdroplets on a matrix on which a biological sample such as cDNA is coated, and different types of samples are arranged in an array. Thus, simultaneous multi-item processing can be performed.

The following can be cited as combinations of the first sample fixed on the substrate in advance and the second and subsequent samples to be reacted with the first sample.

Hereinafter, specific examples of a matrix composed of defined regions on a substrate which can be used in the present invention will be described. (Shape of matrix to which biological sample is bound) The shape of the matrix pattern is not particularly limited, and any shape is possible. However, it is considered from the viewpoint of convenience when supplying a sample onto a prepared substrate. And a shape such as linear (linear), square, or rectangular is preferable in that it can respond to any sample supply. Of course, no problem occurs even if the shape is circular or elliptical.

As a material to be fixed to the substrate as the first sample, unknown base sequences derived from organisms, cDNA libraries, mRNA libraries, plural types of DNAs and RNs
A set, known synthetic or biological DNA or R
NA or a set thereof, a cloned oncogene fragment, a protein fraction derived from an organism containing at least one protein, a single protein, a mixture of known heterologous proteins, a chemical substance, and the like can be mentioned.

(Density of matrix to which biological sample is bound)
The density of the matrix is not particularly limited, but is preferably 400 or less per cm ² . 400 / cm ² is when the shape is square,
The size of one matrix is 500 μm square. When the samples arranged as spots on the array are arranged as spots having a diameter of 100 μm, there are 5 spots in each of the vertical and horizontal directions, that is, a total of 25 spots. In addition, the diameter of the sample solution is set to 20.
In the case of μm, the number of spots that can be arranged in a line is 2
5 and a total of 625 spots can be arranged.

(Preparation of Substrate to which Biological Sample is Bound) Examples of biological samples (biological samples) include nucleic acids and proteins. As the nucleic acid, for example, mRNA, cDN
There is A, as a method of binding these on the substrate, a method of applying a nucleic acid extracted and purified in advance and fixing by adsorption or electrostatic binding, and a functional group on the substrate using an amino group possessed by the nucleic acid And fixing by covalent bonding by the chemical reaction of

The method utilizing negative charge of DNA is a method of electrostatically binding to a solid support surface-treated with a polycation such as polylysine, polyethyleneimine or polyalkylamine, and then blocking excess cations. so,
Commonly used.

(Types of functional groups of solid phase and nucleic acid) Examples of combinations of functional groups used for immobilization include an epoxy group (on a solid phase) and an amino group (terminal of a nucleic acid probe or amino group in a base). And the like. The introduction of an epoxy group to the solid phase surface is performed, for example, by applying polyglycidyl methacrylate having an epoxy group to a solid phase surface made of a resin, or applying a silane coupling agent having an epoxy group to a glass solid phase surface. And the like.

(Binding of Solid Phase to Protein) As a method for binding a protein to a substrate, a method using adsorption similar to nucleic acid and a method using electrostatic binding are exemplified. Further, as a covalent bonding method, a method using an SH group of a cysteine residue may be used in addition to the method using the amino group.

(Method of establishing a protein trade using a thiol group) As a method of using a cysteine residue for immobilizing a protein, there is an example in which a combination of a maleimide group and a thiol group (-SH) is used. That is, by treating the solid phase surface with a maleimide group, the thiol group of the cysteine residue of the protein supplied to the solid phase surface reacts with the maleimide group on the solid phase surface to immobilize the protein. can do.

Various methods can be used to introduce a maleimide group on the surface of the solid phase. For example, a glass substrate is reacted with an aminosilane coupling agent, and then the amino group is reacted with N- ( 6-Maleimidocaproyloxy) succinimide (N- (6-Maleimid
It is possible to react with a reagent (EMCS reagent: manufactured by Dojin) containing ocaproyloxy) succinimide. (Configuration of DNA Matrix Consisting of Hydrophobic Matrix) A further form of immobilizing a biological sample is to form, in advance, a well formed of a hydrophilic and hydrophobic matrix on the solid surface to prevent the connection between spots. A method of providing a DNA probe into the well and performing a binding reaction can be used.

(Material of Matrix / Well) When a probe solution is put on a partitioned matrix to perform a binding reaction, the well constituting portion is hydrophilic and corresponds to the wall surface of the well and a partition from the adjacent well. It is preferable that the portion has a surface made of a material having low affinity for the probe solution. By such a process, even when a slight displacement occurs when the probe solution is supplied to the well, the probe solution can be smoothly supplied to the desired well.

FIG. 2 shows an example of a matrix according to this embodiment. FIG. 2A is a plan view, and FIG. 2B is a BB sectional view thereof. This matrix has a horizontal structure in which a matrix pattern 125 having a frame structure in which concave portions (wells) 127 arranged in a solid phase 103 are formed.
The wells 127 separated from each other by the matrix 125 (convex portion) are provided as through holes (cut-out portions) in the matrix pattern, and the side surfaces thereof are composed of convex portions. Is in an exposed state. The exposed portion of the surface of the solid phase 103 forms a surface that can be bonded to the probe, and the probe is fixed in a predetermined concave portion.

Examples of the material for forming the matrix pattern include metals (chromium, aluminum, gold, etc.) and resins. Resins such as acrylic, polycarbonate, polystyrene, polyimide, acrylic acid monomer, urethane acrylate, and the like, and photosensitive resins such as photoresists containing a black dye or a black content may be used. As specific examples of the photosensitive resin, for example, a UV resist, a DEEP-UV resist, an ultraviolet curable resin, or the like can be used. Examples of the UV resist include a negative resist such as a cyclized polyisoprene-aromatic pisazide-based resist, a phenol resin-aromatic azide compound-based resist, and a positive resist such as a novolak resin-diazonaphthoquinone-based resist.

As the DEEP-UV resist, as a positive resist, for example, polymethyl methacrylate,
Polymethylene sulfone, polyhexafluorobutyl methacrylate, polymethyl isopropenyl ketone, and
A radiation-dispersion type polymer resist such as poly 1-trimethylsilylpropyne bromide, and a dissolution inhibitor-based resist such as o-nitrobenzyl cholate can be mentioned. As a negative resist, polyvinylvinylphenol-3-3 ′ is used.
-Diazidodiphenylsulfone and polyglycidyl methacrylate.

The UV-curable resin contains about 2 to 10% by weight of one or two or more photopolymerization initiators selected from benzophenone and its substituted derivatives, and oxime compounds such as benzyl. And polyester acrylate, epoxy acrylate, and urethane diacrylate.

In order to suppress reflection at the time of detection by the material forming the matrix, it is effective to use a light-shielding material as the material forming the matrix pattern. To this end, it is effective to add a black pigment to the resin, and as the black pigment, carbon black or a black organic pigment can be used.

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

Matrix thickness (height from solid surface)
Is determined in consideration of the method of forming the matrix pattern and the capacity of the well, but is preferably 1 to 20 μm. In particular, it is considered to be a thickness region that effectively prevents cross contamination when a probe solution is supplied to each well by an inkjet method. (Type of sample to be spotted) Examples of the sample spotted as droplets on the biological sample matrix described above include chemical substances such as probe nucleic acids, proteins, and drugs.

The probe nucleic acid is not particularly limited as long as it has a nucleic acid base, such as ribonucleic acid and peptide nucleic acid, in addition to deoxyribonucleic acid. The length of the oligonucleotide probe is not particularly limited, but is 10 m in order to perform an accurate hybridization reaction with cDNA.
It is preferably from er to 50 mer.

The protein utilizes its own fluorescence to produce DN
A-binding protein can be detected.

Some chemical substances can be detected by their own fluorescence. (Method for Preparing Sample Array) As a method for spotting a sample solution at a predetermined position with a size of several tens of microns to 100 microns, there are a pin method, an ink jet method, and a capillary method.

In the pin method, a sample is brought into contact with the tip of a pin, for example, by contacting the tip of the pin with the liquid surface of a solution containing the sample, and then attaching the tip to a solid phase. This is a method for producing In the capillary method using a capillary, the sample solution once sucked into the capillary is mechanically brought into contact with a solid phase from the tip of the capillary similarly to the pin method, thereby supplying the sample solution in an array. For such a spotting operation, each device sold by each company can be used. These methods are considered to be the most preferable methods in that any sample DNA can be supplied. However, DN
A problem may remain in terms of quantitativeness that the viscosity differs depending on the length and concentration of A. Regarding protein trade, these methods are preferable because they are not affected by the size and viscosity of molecules and can supply samples without physical stimulus such as shearing force as in the ink jet method. . (Outline of Sample Array Production Method by Inkjet Method) Examples of samples that can be ejected by the inkjet method include nucleic acids, proteins, and chemicals.

In the ink-jet method, since a shearing force acts, the length of the nucleic acid which can be ejected and the size of the protein are often limited. However, its quantification is other pin method,
It is superior to the capillary method, and is more preferably used for discharging a chemical substance than other methods. The nucleic acid that can be ejected has a base pair length of 1 kb or less, and the protein trade is limited to 100 K daltons or less. Any chemicals can be discharged.

The ejection liquid used to eject and supply the sample by the ink jet can be ejected from the ink jet, and the liquid ejected from the head lands at a desired position, and is mixed with the nucleic acid probe. Any liquid can be used as long as the nucleic acid probe is not damaged during ejection.

From the viewpoint of the ink jet, in particular, the ejection property from a bubble jet (registered trademark) head,
As a characteristic of the liquid, for example, its viscosity is 1 to 15 cp.
s, the surface tension is preferably 30 dyn / cm or more. Further, the viscosity is 1 to 5 cps, and the surface tension is 30 to 50 dyn.
/ Cm, the landing position on the solid phase becomes extremely accurate, and is particularly preferably used.

Therefore, in consideration of the stability of the nucleic acid at the time of ejection, for example, a nucleic acid probe of 2 to 100 mer, particularly 2 to 60 mer, is contained in the solution in an amount of 0.05 to 500 μm.
M, particularly preferably at a concentration of 2 to 50 μM.

FIG. 3 is a schematic explanatory view of a sample solution discharging method by a bubble jet method according to one embodiment of the present invention.
In FIG. 3, reference numeral 101 denotes a liquid supply system (nozzle) which holds a solution containing a sample as a discharge liquid so as to be able to discharge the liquid;
Reference numeral 105 denotes a solid phase to which a nucleic acid probe to be reacted with the sample is bound, and reference numeral 105 denotes a kind of an ink jet head, which is a bubble jet head having a function of applying thermal energy to the liquid and discharging the liquid. Reference numeral 104 denotes a liquid containing a specimen discharged from the bubble jet head. FIG. 4 is a sectional view taken along line AA of the bubble jet head 105 of FIG. 3. In FIG. 4, reference numeral 105 denotes a bubble jet head, 107 denotes a liquid containing a sample solution to be discharged, and 117 denotes discharge energy to the liquid. This is a substrate portion having a heating section to be applied. The substrate portion 117 includes a protective film 109 made of silicon oxide or the like, electrodes 111-1 and 111-2 made of aluminum or the like, a heating resistor layer 113 made of nichrome or the like, and a heat storage layer 115.
And a support body 116 formed of alumina or the like having good heat dissipation properties.

The liquid 107 containing the specimen is formed at the discharge orifice (discharge port) 119 and forms a meniscus 121 by a predetermined pressure. Here, the electrode 111-
When an electric signal is applied to 1,111-2, a region (foaming region) indicated by 123 rapidly generates heat, and the liquid 107 in contact therewith is discharged and flies toward the surface of the solid phase 103. The amount of liquid that can be ejected using a bubble jet head having such a structure depends on the size of the nozzle and the like, but can be controlled to, for example, about 4 to 50 picoliters, and the sample probe can be densely arranged. This is extremely effective as means for arranging.

[0062] From the viewpoint of the ink jet, particularly from the viewpoint of dischargeability from a bubble jet head, the liquid preferably has, for example, a viscosity of 1 to 15 cps and a surface tension of 30 dyn / cm or more. In addition, the viscosity is 1 to
When the surface tension is 5 cps and the surface tension is 30 to 50 dyn / cm, the landing position on the solid phase becomes extremely accurate, and it is particularly preferably used.

Therefore, in consideration of the stability of the nucleic acid at the time of ejection, for example, 100 to 10000 meme in the solution.
r, especially a nucleic acid probe of 100-5000 mer,
It is preferable to contain it at a concentration of 10 μM or less, particularly 1 μM or less.

The composition of the ejection liquid is such that it does not substantially affect the nucleic acid probe when mixed with the nucleic acid probe as described above and when ejected from the ink jet, and when the ink jet is used. The liquid composition is not particularly limited as long as it satisfies the conditions that enable normal ejection to the solid phase, for example, glycerin,
A liquid containing urea, thiodiglycol or ethylene glycol, isopropyl alcohol and acetylene alcohol represented by the following formula is preferred.

[0065]

Embedded image

(In the above formula (I), R ₁ , R ₂ , R ₃ and R ₄
Represents an alkyl group, specifically, for example, a linear or branched alkyl group having 1 to 4 carbon atoms, m and n each represent an integer, and m = 0, n = 0, or 1 ≦ m + n ≦ 30
And when m + n = 1, m or n is 0. More specifically, urea is 5 to 10% by weight (wt), glycerin is 5 to 10% by weight, and thiodiglycol is 5 to 10%.
wt%, and 0.02 to 5 wt% of acetylene alcohol represented by the above formula (I), more preferably 0.5 to 1 wt%.
A liquid containing wt% is preferably used.

[0067]

The present invention will be described below in detail with reference to examples. Example 1 p53 on a sample matrix substrate divided by a pattern
A glass substrate with a black matrix for a sample matrix is prepared.

1. Preparation of black matrix introduction substrate coated with polylysine Glass substrate made of synthetic quartz (60
mm × 50 mm) is ultrasonically cleaned using a 2% aqueous sodium hydroxide solution, and then subjected to UV ozone treatment to clean the surface. Next, a polylysine solution (manufactured by Sigma) is applied to the entire surface by a spin coater. Furthermore, a DEEP-UV resist containing carbon black (a negative type resist for a black matrix) (trade name: BK-7
39P (manufactured by Nippon Steel Chemical Co., Ltd.) is applied by a spin coater so that the film thickness after curing becomes 5 μm, and the substrate is cured by heating at 80 ° C. for 5 minutes on a hot plate. D
The rejection (X) between adjacent wells in FIG. 1 was 100 μm in a 1 cm × 1 cm area using an EEP-UV exposure apparatus.
m, and proximity exposure using a mask patterned so that the shape of the well becomes a square of 1 mm × 1 mm, then developing with a developer of an inorganic alkali aqueous solution using a spin dryer, and further with pure water After washing, the developer is completely removed.

Next, it is easily dried using a spin dryer,
After that, the resist is fully cured by heating at 180 ° C. for 30 minutes in a clean oven, and the wells are arranged in a predetermined arrangement to form 400 wells.
A substrate is obtained in which the adjacent wells are separated by a black matrix. The volume of each well is 5
Assuming μm, 5 μl is calculated.

2. Immobilization of sample DNA (1) Preparation of cDNA library The p53 gene is obtained by PCR from 64 types of cDNA libraries obtained from cancer tissues.

That is, Catrimox-14 (Biotechnology) was obtained from each of the fibers collected by biopsy.
y company) was used to obtain an RNA sample. Based on this sample solution, First-Strand cDNA Sy
nthesis Kit (Life Sciences)
To obtain a cDNA library. (2) Amplification of p53 gene having T3 binding site by PCR method Based on the cDNA library, CLONTECH “H
PCR reaction is performed using "human p53 Amplimer Set".

As a PCR reaction solution, "one shop
tLA PCR Mix "(Takara Shuzo) was used. PC
The composition of the R reaction solution was as follows: one shot LA PCR Mix 25 μl 5 ′ primer (20 μM) 1 3 ′ primer (20 μM) 1 cDNA library solution 1 DW 22/50 μl The PCR cycle was heat denaturation at 95 ° C. for 5 minutes, followed by 95 ° C. for 5 minutes. 3
A cycle of 0 second, 55 ° C. for 30 seconds and 72 ° C. for 60 seconds is performed 29 times, and finally, the reaction is carried out at 72 ° C. for 5 minutes and stored at 4 ° C.

After the reaction, gel electrophoresis was performed, and 300 me
Confirm that there is a product in the molecular weight range of about r.
oSpin Column S200 (Pharmac
Purification in ia) yields the p53 gene (p53 DNA). (3) Synthesis of single-stranded p53 DNA Single-stranded labeled DN by a PCR reaction using the DNA obtained in (2) above as a template and 5 ′ primer (Takara Shuzo)
Get A. The reaction solution was one shot LA PCR Mix 25 μl 5 ′ primer (20 μM) 1 P53 DNA 1 DW 23/5
At 0 μl, the reaction cycle is 30 seconds at 96 ° C. and 15 seconds at 60 ° C.
Repeat for 24 minutes at 60 ° C. for 24 seconds and finally store at 4 ° C. Then, MicroSpin Column S20
Purify at 0. (4) Immobilization of p53 cDNA 5 μl of the single-stranded DNA obtained in (3) above was injected into each well of the polylysine-coated substrate with a black matrix prepared in (1) above under a microscope, and fixed by electrostatic binding. I do.

3. P by oligonucleotide probe
Analysis of 53 gene mutations 64 kinds of DNAs were selected by paying attention to the 248th and 249th amino acid sequences of p53 gene, which is a tumor suppressor gene. That is, in the nucleotide sequence CGGAGGG, the mutation frequency is high because the first C mutates to T, the second A to G, and the third G of the sequence corresponding to the 249th amino acid to T. It is known to be the case. Thus, 64 types of probes were designed by focusing on these three base sequences.

That is, the total length of the probe is 18 mer,
It has a structure in which 6 bases containing this mutation are located in the middle, and before and after that are sandwiched by a common sequence. The consensus sequence is ATGAAC from the 5 'end, and the portion containing the subsequent mutation is NN
GAGN followed by the common part is CCCATC, and the final sequence is ^{5 '} ATGAACNNGAGNCC
CATC ^{3 '} . Here, the portion represented by N corresponds to four types of nucleic acid bases, A, G, C, and T. Probe DN
Since A is a sequence complementary to the sequence to be detected (the above sequence), actually, ^{5 ′} MGGGNCCTCNNGTTTCA
T3 ^' . Rhodamine is bound to the 5 'end of each probe sequence and labeled. Specific base sequences of these 64 types of labeled DNA probes are as shown in Table 1 below.

[0076]

[Table 1]

Next, each of the 64 kinds of labeled probe DNAs is prepared into an 8 μM solution containing glycerin, urea and thiodiglycol at a final concentration of 7.5% and acetylenol EH at a final concentration of 1%. Head B for BJ printer
Each of six nozzles of C62 (manufactured by Canon Inc.) was filled with 100 μl of a different probe solution. Using two heads so that six types of DNA can be ejected per head, 12 types of DNA are ejected at a time, and the heads are replaced six times, so that spots of 64 types of DNA are independently formed. To be discharged. In this way, a total of 64 types of probes are discharged in an 8 × 8 array into each well of the black matrix coated with polylysine.

FIG. 5 is a diagram showing the arrangement of 64 types of DNA probes to be ejected on each black matrix. In this case, 64 types of DNA probes are implanted in one matrix.

Thereafter, the substrate on which each probe was spotted was left in a humidification chamber set at 40 ° C.
Perform a hybridization reaction.

Thereafter, the substrate is washed with a 10 mM phosphate buffer containing 100 mM NaCl to remove DNA probes which have not been involved in the formation of the hybrid.

The DNA array after the hybridization reaction is observed using an inverted fluorescence microscope equipped with a filter set suitable for rhodamine.

If the gene as the specimen has a normal base sequence, a spot with the highest fluorescence intensity should be observed at the position of the 42nd DNA probe relative to the gene. These are considered to be derived from a hybrid of the probe DNA and the p53 gene having a normal sequence amplified by PCR. In the case of a sample gene having a mutation, a detectable spot is found at a position other than No. 42, and the sequence mutated can be known from the sequence of the DNA probe supplied at that position.

Example 2 (Evaluation of the Presence or Absence of an Oncogene Using mRNA) Extraction of mRNA "QuickPre" from cancer tissues collected by biopsy
p Micro mRNA Purification
Kit (Amersham Pharmacia b
The mRNA is extracted using iotech). This mRNA is bound to a polylysine substrate with a black matrix as in Example 1.

2. Inspection of presence / absence and types of oncogenes using various oncogene probe arrays A set of cloned oncogenes (18 species, manufactured by Takara Shuzo Co., Ltd.) was purchased, and “LabelITnon-RI L
Rhodamine labeling is performed using the "Abeling Kits".

The labeled 18 oncogene probes were placed on the substrate to which the above mRNA was bound, Cartesia
Spots are arranged in a 4 × 5 array using a microarray manufacturing device (pin method) manufactured by n Technologies.

Further, a hybridization reaction is carried out in the same manner as in Example 1.

[0087] The type of oncogene present in the mRNA fraction extracted from each tissue can be known.

At this time, detection is possible with one type of label regardless of the type of oncogene.

[0089]

According to the present invention, it is possible to provide a method for simultaneously testing multiple specimens in multiple items.

[0090]

[Sequence List] SEQUENCE LISTING <110> Canon INC. <120> An assay of many samples for multiple items at the same time <130> 3912041 <160> 64 <210> 1 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 1 gatgggactc aagtt cat <210> 2 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 2 gatgggactc aggtt cat < 210> 3 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 3 gatgggactc acgtt cat <210> 4 <211> 18 <212> DNA <213> Artificial sequence <220 > <223> Sample origonucleotide <400> 4 gatgggactc atgtt cat <210> 5 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 5 gatgggactc gagtt cat <210> 6 < 211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 6 gatgg gactc gggtt cat <210> 7 <211> 18 <212> DNA <213> Artificial sequence <220> <223 > Sample origonucleotide <400> 7 gatgggactc gcgttcat <210> 8 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400 > 8 gatgggactc gtgttcat <210> 9 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 9 gatgggactc cagttcat <210> 10 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 10 gatgggactc cggttcat <210> 11 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 11 gatgggactc ccgttcat <210> 12 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 12 gatgggactc ctgttcat <210> 13 <211> 18 <212> DNA <213> Artificial sequence <220> <223 > Sample origonucleotide <400> 13 gatgggactc tagttcat <210> 14 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 14 gatgggactc tggttcat <210> 15 <211> 18 <212 > DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 15 gatgggactc tcgttcat <210> 16 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 16 gatgggactct tgttcat <210> 17 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 17 gatggggctc aagttcat <210> 18 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 18 gatggggctc aggttcat <210> 19 <211> 18 <212> DNA < 213> Artificial sequence <220> <223> Sample origonucleotide <400> 19 gatggggctca cgttcat <210> 20 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 20 gatggggctc atgttcat < 210> 21 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 21 gatggggctcg agttcat <210> 22 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 22 gatggggctc gggttcat <210> 23 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 23 gatggggctc gcgttcat <210> 24 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 24 gatggggctc gtgttcat <210> 25 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400 > 25 gatggggctc cagttcat <210> 26 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonu cleotide <400> 26 gatggggctc cggttcat <210> 27 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 27 gatggggctc ccgttcat <210> 28 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 28 gatggggctc ctgttcat <210> 29 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 29 gatggggctct agttcat <210> 30 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 30 gatggggctc tggttcat <210> 31 <211> 18 <212> DNA <213> Artificial sequence <220 > <223> Sample origonucleotide <400> 31 gatggggctc tcgttcat <210> 32 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 32 gatggggctc ttgttcat <210> 33 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 33 gatgggcctc aagttcat <210> 34 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide < 400> 34 gatgggcctc aggttcat <210> 35 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 35 gatgggcctc acgttcat <210> 36 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 36 gatgggcctc atgttcat <210> 37 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 37 gatgggcctc gagttcat <210> 38 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 38 gatgggcctc gggttcat <210> 39 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 39 gatgggcctc gcgttcat <210> 40 <211> 18 <212> DNA <213> Artificial sequence <220 > <223> Sample origonucleotide <400> 40 gatgggcctc gtgttcat <210> 41 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 41 gatgggcctc cagttcat <210> 42 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 42 gatgggcctc cggttcat <210> 43 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide < 400> 43 gatgggcctc ccgttcat <210> 44 <211> 18 <212> DNA <213> Artificial sequence <220> <22 3> Sample origonucleotide <400> 44 gatgggcctc ctgttcat <210> 45 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 45 gatgggcctc tagttcat <210> 46 <211> 18 < 212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 46 gatgggcctc tggttcat <210> 47 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 47 gatgggcctc tcgttcat <210> 48 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 48 gatgggcctc ttgttcat <210> 49 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 49 gatgggtctc aagttcat <210> 50 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 50 gatgggtctc aggttcat <210> 51 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 51 gatgggtctc acgttcat <210> 52 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 52 gatgggtctc atgttcat <210> 53 <211> 18 <212> DNA <213> Artificial sequence < 220> <223> Sample origonucleotide <400> 53 gatgggtctc gagttcat <210> 54 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 54 gatgggtctc gggttcat <210> 55 <211 > 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 55 gatgggtctc gcgttcat <210> 56 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 56 gatgggtctc gtgttcat <210> 57 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 57 gatgggtctc cagttcat <210> 58 <211> 18 <212> DNA < 213> Artificial sequence <220> <223> Sample origonucleotide <400> 58 gatgggtctc cggttcat <210> 59 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 59 gatgggtctc ccgttcat < 210> 60 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 60 gatgggtctc ctgttcat <210> 61 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 61 gatgggtctc tagttcat <210> 62 <211> 18 <212> DNA <213> Artificial se quence <220> <223> Sample origonucleotide <400> 62 gatgggtctc tggttcat <210> 63 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 63 gatgggtctc tcgttcat <210> 64 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Sample origonucleotide <400> 64 gatgggtctc ttgttcat

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of an arrangement of a defined area on a substrate according to the present embodiment.

FIG. 2 shows an example of a matrix in this embodiment, and FIG.
2A is a plan view, and FIG. 2B is a BB cross-sectional view thereof.

FIG. 3 is a schematic explanatory view of a sample solution discharging method by a bubble jet method according to an embodiment of the present invention.

FIG. 4 is a sectional view of the bubble jet head 105 taken along line AA of FIG. 3;

FIG. 5 is a layout diagram of ejected 64 types of DNA probes on each black matrix.

[Explanation of symbols]

 Reference Signs List 101 Liquid supply system (nozzle) 103 Solid phase 104 Liquid 105 Bubble jet head 107 Liquid 109 Protective film 111-1, 111-2 Electrode 113 Heating resistor layer 115 Heat storage layer 116 Support 117 Substrate unit 119 Discharge port (orifice) 121 Meniscus 121 123 Foaming area

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 35/02 ZCC G01N 37/00 102 35/10 103 37/00 102 C12N 15/00 ZNAA 103 G01N 35 / 06 A (72) Inventor Tomohiro Suzuki 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Inventor Eiji Shimizu 3-30-2, Shimomaruko 3-chome, Ota-ku, Tokyo F-term in Canon Inc. (Reference) 2G042 AA01 BD19 2G058 CC02 CC08 EA11 EB00 ED02 FB02 GA02 4B024 AA11 AA20 CA09 HA14 HA20 4B063 QA01 QA13 QA18 QQ02 QQ04 QQ05 QQ20 QQ42 QQ52 QR32 QR35 QR55 QR82 QS34

Claims (61)

[Claims] 1. A method for simultaneously assaying the reactivity of a first sample and a plurality of samples, wherein the first sample is located within a defined area on a substrate which is pre-bonded over the entire surface. The second to second + nth (n ≧ 1) samples having different properties are independently arranged as regions smaller than the defined region, and the first sample and the second to second + n A method for testing reactivity between a reagent and a specimen, wherein the reactivity between each of the samples (n ≧ 1) is assayed. 2. The inspection method according to claim 1, wherein the first sample is a biological sample, and the second and subsequent samples are samples whose properties are already clarified. 3. The inspection method according to claim 2, wherein the first sample is a biological sample, and the second and subsequent samples are synthesized. 4. The first sample is a nucleic acid of unknown base sequence derived from an organism, and the second and subsequent samples are synthetic nucleic acids with known sequences, and their binding is examined based on their complementarity as their reactivity. The inspection method according to claim 3. 5. The method according to claim 1, wherein said first sample is an mR extracted from an organism.
The inspection method according to claim 4, wherein the inspection method is a set of NAs. 6. The mR extracted from an organism as the first sample.
The test method according to claim 4, which is a cDNA library synthesized based on NA. 7. The method according to claim 2, wherein said first sample is a nucleic acid of unknown base sequence derived from a living organism, and said second and subsequent samples are synthesized chemical substances, and their binding is examined as their reactivity. Inspection method described in 1. 8. The method according to claim 2, wherein the first sample is a nucleic acid of unknown base sequence derived from a living organism, the second and subsequent purified proteins, and their binding is examined as their reactivity.
Inspection method described in 1. 9. The inspection method according to claim 1, wherein the first sample has already been characterized, and the second and subsequent samples are biological samples. 10. The test method according to claim 1, wherein the first sample is a gene sequence whose properties have been clarified, and the second and subsequent samples are biological samples. 11. The test method according to claim 1, wherein the first sample is a cloned oncogene fragment, and the second and subsequent samples are nucleic acid samples derived from an organism. 12. The method according to claim 1, wherein the first sample is a protein fraction extracted from an organism, and the second and subsequent samples are purified single species of protein, and their binding is examined as their reactivity. 2. The inspection method according to 1. 13. The method according to claim 13, wherein said first sample is a purified single species of protein, and said second and subsequent samples are protein fractions extracted from an organism. The inspection method according to claim 1, wherein: 14. The method according to claim 2, wherein the first sample is a protein fraction extracted from an organism, and the second and subsequent samples are chemically synthesized chemical substances, and their binding is examined as their reactivity. Inspection method described in 1. 15. The method according to claim 2, wherein the first sample is a purified single kind of protein, the second and subsequent samples are chemically synthesized chemical substances, and their binding is examined as their reactivity. Inspection method described in 1. 16. The method according to claim 1, wherein the first sample is a chemically synthesized chemical substance, and the second and subsequent samples are nucleic acids extracted from an organism, and their binding is examined as their reactivity. Inspection method. 17. The method according to claim 1, wherein the first sample is a chemically synthesized chemical substance, the second and subsequent samples are protein fractions extracted from an organism, and their binding is examined as their reactivity. 1. The inspection method according to 1. 18. The method according to claim 1, wherein the first samples having different properties are respectively bonded to different regions over the entire surface, and a plurality of the bonded regions are present in a matrix on the substrate. Inspection method described. 19. The method according to claim 1, wherein the first sample is a nucleic acid derived from a different species, tissue or cell.
9. The inspection method according to 8. 20. The test method according to claim 18, wherein the first sample is a protein extracted from different species, tissues or cells. 21. The density of a matrix formed by the binding regions of the first sample having different properties is 400
19. The inspection method according to claim 18, wherein the density is not more than / cm ² . 22. A spot array composed of spots of second, third and subsequent samples is arranged in the same arrangement in each matrix on a matrix formed by the binding regions of the first sample having different properties. Item 19. The inspection method according to Item 18. 23. The inspection method according to claim 1, wherein the substrate is glass. 24. The inspection method according to claim 1, wherein the first sample is fixed on a substrate by electrostatic coupling. 25. The inspection method according to claim 1, wherein the first sample is fixed on a substrate by a covalent bond. 26. The inspection method according to claim 25, wherein the first sample is bonded to the glass substrate by a chemical reaction between a maleimide group introduced on the surface of the glass substrate and a thiol group of the sample. 27. The test method according to claim 26, wherein the first sample is a protein, and the thiol group is due to a cysteine residue in the protein. 28. After the maleimide group introduces an amino group on the glass surface, the amino group and succiimidyl-4-
The inspection method according to claim 26, which is introduced by reacting with (maleimidophenyl) butyrate. 29. The inspection method according to claim 26, wherein the first sample is formed by a chemical reaction between an ethoxy group introduced on the surface of the glass substrate and an amino group of the first sample. 30. The method according to claim 29, wherein the amino group is an amino group present in a nucleic acid base. 31. The substrate surface is previously partitioned into matrices, and each of these matrices has a first surface.
The test method according to claim 1, wherein a biological sample having a different sex between the matrices is previously bound as the second sample. 32. The inspection method according to claim 31, wherein a wall portion defining the matrix is hydrophobic, and a bottom portion in the matrix is hydrophilic. 33. The wall thickness of the matrix is 1 to 20.
The inspection method according to claim 32, wherein the diameter is μm. 34. The inspection method according to claim 1, wherein the diameter of the spot formed by the second and subsequent sample solutions formed in the defined area to which the first sample is bonded is 200 μm or less. 35. The inspection method according to claim 1, wherein the density of spots formed by the second and subsequent sample solutions formed in the defined region to which the first sample is bonded is 400 / cm ² or more. 36. The inspection method according to claim 1, wherein the second and subsequent samples are supplied by an inkjet method. 37. The second and subsequent samples supplied by the ink jet method are nucleic acids, and have a base pair length of 2 to 2.
37. The method of claim 36, which is 100 base pairs long. 38. The test method according to claim 37, wherein the nucleic acid supplied by the inkjet method is supplied as an aqueous solution, and the concentration of the aqueous solution is 0.05 μM to 600 μM. 39. The inspection method according to claim 36, wherein the ink jet method is a bubble jet method. 40. The second and subsequent samples are supplied by physically contacting the sample solution with the substrate after the pins contact the liquid surface of the reservoir for the sample solution. The inspection method according to claim 1. 41. The supply of the second and subsequent samples is performed by physically contacting the substrate at the tip of the capillary after the solution of the sample is sucked from the sample supply unit using a capillary. 2. The inspection method according to 1. 42. A biological sample matrix, wherein a plurality of types of biological samples having different origins are present on respective matrices partitioned on a substrate. 43. The DNA matrix according to claim 42, wherein the biological sample is a cloned oncogene fragment. 44. The biological sample according to claim 4, wherein the biological sample is mRNA.
3. The mRNA matrix according to 2. 45. The biological sample according to claim 4, wherein the biological sample is cDNA.
3. The cDNA matrix according to 2. 46. The cDNA matrix according to claim 42, wherein said biological sample is a cDNA library. 47. The protein matrix according to claim 42, wherein said biological sample is a plurality of proteins each having a different three-dimensional structure. 48. The biological sample matrix according to claim 42, wherein the density of the matrix to which the biological sample is bound is 400 / cm ² or less. 49. The biological sample matrix according to claim 42, wherein the substrate is glass. 50. The biological sample matrix according to claim 42, wherein the biological sample is fixed on a substrate by electrostatic coupling. 51. The biological sample matrix according to claim 42, wherein the biological sample is immobilized on a substrate by a covalent bond. 52. The biological sample matrix according to claim 51, wherein the biological sample is bonded to the glass substrate by a chemical reaction between a maleimide group introduced on the surface of the glass substrate and a thiol group of the biological sample. 53. The biological sample according to claim 52, wherein the biological sample is a protein, and these are bound by a chemical reaction between a maleimide group introduced on the surface of the glass substrate and a thiol group of a cysteine residue of the protein. Matrix. 54. After the maleimide group introduces an amino group to the glass surface, the amino group and succiimidyl-4-
53. The biological sample matrix according to claim 52, wherein the matrix is introduced by reacting with (maleimidophenyl) butyrate. 55. The biological sample matrix according to claim 51, wherein the biological sample is a nucleic acid, and these are bound by a chemical reaction between an epoxy group introduced on the surface of the glass substrate and an amino group of the nucleic acid. 56. The biological sample matrix according to claim 42, wherein the plurality of types of biological samples are supplied on respective matrices separated on a substrate by an inkjet method. 57. The biological sample matrix according to claim 42, wherein the substrate surface is previously partitioned into a matrix, and a plurality of types of biological samples having different origins are respectively bonded to respective regions of the matrix in advance. 58. The biological sample matrix according to claim 57, wherein a wall portion defining the matrix is hydrophobic, and a bottom portion of the matrix is hydrophilic. 59. A thickness of a wall portion of the matrix is from 1 to 1.
59. The biological sample matrix according to claim 58, which is 20 [mu] m. 60. The biological sample matrix according to claim 57, wherein the plurality of kinds of oligonucleotides are supplied to wells of a partitioned matrix by an inkjet method. 61. The biological sample matrix according to claim 56, wherein the ink jet method is a bubble jet method.