JPH1199000A - Reaction field array, method for producing reaction field array, reaction method using reaction field array, and method for quantifying substances in sample solution using reaction field array - Google Patents

Abstract

(57) [Summary] [Problem] It is difficult to confirm the synthesis of a probe when using sequential synthesis of a probe, and further, it is not possible to remove a by-product
It solves the problems in the prior art that cross-contamination occurs when supplying a reaction species including a probe and that the area of an individual reaction region cannot be made sufficiently small. To provide a reaction field array which can detect optically with high sensitivity even when the area is minute. SOLUTION: When manufacturing a reaction field array in which a plurality of reaction fields for reacting two or more kinds of substances in a liquid medium are arranged separately from each other, a surface of a substrate having affinity for the liquid medium. A matrix pattern having a predetermined arrangement of reaction fields consisting of concave portions which are separated from each other by convex portions and whose bottom surface is partially exposed on the bottom surface, and whose convex surface is incompatible with a liquid medium. To obtain a reaction field array.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention is the screening of drugs such as a therapeutic agent BACKGROUND OF THE INVENTION The gene fingerprinting, determination of the base sequence of the gene by hybridization (SBH: S equencing B y H ybrid
reaction field array provided with a plurality of reaction fields, which can be used for so-called combinatorial chemistry, which handles a small amount of multiple reactions at the same time, which is used for simultaneous multi-item detection of test substances, etc. A reaction method using the reaction field array and a method for quantifying substances in a sample solution using the reaction field array.

[0002]

2. Description of the Related Art In recent years, for screening a drug such as a therapeutic drug, for example, a plurality of oligopeptides which are expected to interact with a drug to be screened are prepared, and the drug containing the drug to be screened is contained. So-called combinatorial chemistry, such as analyzing the interaction with a plurality of drugs and identifying the required drug as a result, has been attracting attention. It is inefficient for blindly designing drugs, and it takes a lot of time and money to evaluate the designed and synthesized drugs by animal experiments, etc., so combinatorial chemistry as an alternative means This is because it has been needed.

As such combinatorial chemistry probes, the above-mentioned oligopeptide probes can be mentioned first. As means for binding such a probe to the surface of a solid phase, latex particles having a functional group capable of binding to the probe on the surface thereof are also commercially available (Calbiochem-Novabiochem).
Company). Also, US Pat. No. 5,143,854 discloses a method for producing an oligopeptide array using a photodegradable protecting group and a photolithography process in combination.

On the other hand, as a method of detecting a target nucleic acid having a specific base sequence by using hybridization of a nucleic acid probe to the target nucleic acid, a conventional so-called Southern hybridization has been used. A method has been proposed in which a plurality of types of nucleic acid probes are arranged, a specimen sample containing a target nucleic acid is hybridized thereto, and detection is performed by one mode of combinatorial chemistry.

For example, Japanese Patent Application Laid-Open No. 3-505157 proposes an apparatus for analyzing a polynucleotide sequence consisting of a complete set of oligonucleotides of a specific length immobilized on a support surface, or a specific portion. Further, US Pat. No. 5,202,231 discloses a method for analyzing a nucleotide sequence of a polynucleotide by such a method (sequence).
quenching by hybridizio
n) has been proposed. Also, U.S. Pat.
Japanese Patent Publication No. 186 discloses a method for producing a nucleic acid probe array on a solid-phase substrate using both a photodegradable protecting group and a photolithography process.

[0006] On the other hand, in simultaneous reaction and detection of multiple items or multiple samples in enzyme immunoassay and the like, it is common to conduct a reaction in a microplate having a maximum of 96 wells and read the results with a microplate reader. However, there is a limit to rapidly processing a large amount of a sample.

[0007]

One of the main aims of combinatorial chemistry is how to efficiently supply a large variety (in number) of reactive species to a reaction field. In other words, how to efficiently supply various types and small amounts (as liquid volumes) to the reaction field without causing cross-contamination between adjacent reaction fields. From such a point of view, the method using the microplate reader described above has a limit in principle, although high-speed, large-scale processing has recently been advanced by robotization. In addition, there is a problem that the volume of liquid handled in one well is relatively large, about 20 μl to 100 μl.

Further, the above-mentioned US Pat. No. 5,143,854
In the method of synthesizing a probe array on a solid phase using a photodegradable protecting group and photolinography described in JP-A Nos. 5,424, 186 and 5,424, 186, various kinds of probes are arranged on a substrate. Is possible, but since each probe is in the same plane, the substance to be reacted with the probe is supplied to the entire probe, so that the type of reaction cannot be changed individually. In addition, since the oligopeptides and oligonucleotides, which are sequentially synthesized on the solid phase as the probe, are used as they are, the synthesis of the probe cannot be confirmed, and in addition, by-products included in the synthesis (the objective (Oligomers shorter than the length) cannot be removed.

As a means for solving these problems, there has been proposed a method of supplying a synthesized and purified reaction species to a reaction field using a microdispenser.

[0010] For example, Khrapko et al. Have introduced a method of preparing a DNA probe array by spotting a DNA solution on a polyacrylamide gel using a micropipette (DNA Suequnc).
ing 1, 675-388, 1991). In this method, a relatively small amount of DNA solution can be supplied, but the area to be supplied cannot be specified. Therefore, there is a problem in quantitativeness. In addition, cross-contamination between adjacent spots at the time of probe supply is difficult. There is also a risk. There is a similar problem when supplying the other reactive species.

Further, the supply amount of the reactive species is made small,
Sequential synthesis of nucleic acid probes using an ink-jet method to enable a large amount of reaction, mainly by a solid-phase porous method, has also been proposed (International Publication WO 95/2511).
6). As described above, this method has a problem of sequentially synthesizing a probe and a problem that a region to be supplied cannot be specified.

Several means have been proposed to solve the problem that the supply region of the reactive species cannot be specified.

For example, Christey et al. Have introduced a method of preparing a DNA probe array by patterning a silane coupling agent having an appropriate functional group applied on a substrate and spotting a DNA probe solution. (Nucleic Acid Research,
Vol. 24, no. 15, 3040-3047, 19
96).

Lemmo et al. Have introduced a means for supplying a reagent substance to each well of a molded polypropylene sheet (plate) having a large number of wells on the surface using a microdispenser (Anal. Ch.).
em. , 69, 543-551, 1997). Here, 8 × 8 wells of polypropylene resin each were placed in a well of a microdispenser by 8 micron dispensers.
A method for supplying about 1 μl of a reagent solution is described.
The size of the well is estimated to be about 3 mm in diameter and about 2 mm in depth, and the size of the molded plate is 8.5.
There is a description of inches × 11 inches. As described above, since the well size that can be actually formed by molding is about several millimeters, the number of wells forming an array is limited to 48 × 48 in consideration of the size of the entire plate. The overall size is also relatively large. In practice, when using combinatorial chemistry, it is desirable that a larger number of types of probe species be present, and that the size of the plate be smaller. In addition, when using a plate molded with the above-mentioned polypropylene, the whole is water-repellent, so it is particularly difficult to supply a commonly used aqueous solution of a biological substance such as nucleic acid into a small well. And may also cause undesirable contamination with the solution in the adjacent well at the same time.

Furthermore, Japanese Patent Publication No. 7-508831 discloses a means for supplying a nucleic acid probe solution to a portion where a silicon substrate is patterned by using a microdispenser. According to this method, it is considered that necessary probe types and array sizes are obtained, but the problem of cross-contamination at the time of supplying a probe or supplying an individual sample still remains.

On the other hand, international patent application WO95 / 35505
In, for example, a nitrocellulose filter lined with a non-water permeable film is partitioned with silicone rubber,
There is disclosed a method in which a DNA probe solution is supplied in the form of an array and a DNA array is prepared by non-covalent bonding.
A method of examining a plurality of specimens while preventing cross-contamination by disposing a plurality of sections partitioned by silicon rubber on a substrate has been disclosed, but specific descriptions regarding individual DNA reaction regions are disclosed. Absent.

An object of the present invention is to solve the above-mentioned problems of the prior art,
That is, it is difficult to confirm the synthesis of the probe when using the sequential synthesis of the probe, furthermore, it is not possible to remove by-products, it is a problem of cross-contamination when supplying the reactive species containing the probe, An object of the present invention is to solve the problem that the area of the region cannot be made sufficiently small.

Another object of the present invention is to provide a technique capable of optically detecting the result of a reaction in a reaction area with high sensitivity even when the reaction area is minute.

[0019]

According to the method for producing a reaction field array of the present invention, a plurality of reaction fields for reacting two or more substances in a liquid medium have a predetermined affinity for the liquid medium. And each of the reaction fields is isolated from each other by a second region having a lower affinity for the liquid medium as compared to the affinity of the first region. A method for producing a reaction field array, wherein the substrate surface having a predetermined affinity for the liquid medium has a lower affinity than the substrate surface having the predetermined affinity for the liquid medium. Forming a convex matrix pattern on the substrate surface, forming the substrate surface exposed corresponding to the matrix pattern as a first region, and defining the matrix pattern as a second region. It is characterized by doing.

The reaction field array of the present invention can be used in a liquid medium.
Having a plurality of reaction fields for reacting more than one kind of substance,
A reaction field array in which respective reaction fields are arranged separately from each other, wherein the reaction field is constituted by a first region having a predetermined affinity for the liquid medium, and the reaction field is It is characterized in that it is isolated from each other by a second region that has a lower affinity for the liquid medium than the first region and that is convex with respect to the first region. .

The method for reacting the substance of the present invention comprises the steps of:
A method for supplying a substance to each reaction field of a reaction field array in which a plurality of reaction fields for reacting at least one kind of substance are arranged separately from each other, and reacting the substance in the reaction field, A reaction field array comprising a first region in which the reaction field exhibits a predetermined affinity for the liquid medium, wherein the reaction field has an affinity for the liquid medium in which the first region exhibits an affinity; It is characterized in that it is isolated from each other by a second region having a lower affinity than the first region and having a lower affinity.

The method for quantifying a substance according to the present invention is a method for quantifying a first substance contained in a sample solution, the method comprising: (a) forming a first region having a predetermined affinity for the sample solution; A plurality of reaction fields, the reaction fields being isolated from each other by a convex second region having a lower affinity for the sample solution as compared to the affinity of the first region. Providing said reaction field array; (b) supplying said sample solution to each reaction field of said reaction field array; (c) reacting said first substance by interaction with said first substance. Supplying a reagent that provides a quantitatively detectable signal to each reaction field of the reaction field array; and (d) quantitatively detecting an interaction between the first substance and the reagent; It is characterized by having.

According to the present invention, for example, a concave portion serving as a reaction field arranged in a matrix on a substrate is exposed on its bottom surface with a substrate surface having an affinity for a liquid medium constituting a reaction system, Further, by forming the surface of the convex portion forming the peripheral wall to be incompatible with the liquid medium, the supply of the solution into the concave portion when supplying the solution containing the reactive substance to the liquid medium is performed. Since the smoothing is performed and the surface of the convex portion is incompatible with the solution, diffusion of the solution on the convex portion is restricted, and cross contamination between adjacent concave portions can be effectively prevented. A good solution supply to the recess can be achieved, for example, up to about several tens of times the volume of the recess.

Further, according to the present invention, a reaction field array having such a function can be manufactured efficiently and with high accuracy.

Since the matrix pattern of the concave portion can be formed by using a fine patterning technique as described later, the area is sufficiently small and a large number of reaction fields are formed.
For example, it can be formed on a 1 cm × 1 cm chip.

In the present invention, in addition to the affinity for the liquid medium itself, the liquid medium has at least one of the substances to be reacted, which is necessary for the reaction, in addition to the affinity for the liquid medium on the substrate surface. Substances, furthermore, reagents and the like in the case of qualitative and quantitative determinations, as well as affinity for the case of containing one or more reaction products, etc. The same is true.

[0027]

FIG. 1A is a plan view of a reaction field array according to one embodiment of the present invention, and FIG. 1B is a cross-sectional view along the line AA. This reaction field array has a plurality of first regions 3 as reaction fields arranged in a matrix on the substrate 1. Each of the first regions has a structure separated from each other by a matrix pattern 2 which is convex with respect to the surface of the first region. First area 3
Indicates that the surface 4 of the substrate 1 is exposed.
Is formed on a surface having an affinity for a liquid medium when two kinds of substances react with each other. On the other hand, the surface of the convex matrix pattern 2 (second region)
Is formed on a surface that is less compatible with the liquid medium than the first region.

Specifically, when the liquid medium constituting the reaction system is an aqueous medium (water or a liquid medium mainly composed of water), the surface of the substrate 1 is made hydrophilic, and the surface of the projections of the matrix pattern is made hydrophilic. Preferably, it is hydrophobic. When the liquid medium is non-aqueous, it is preferable to form the surface of the substrate 1 lipophilic and the surface of the matrix pattern non-lipophilic.

More specifically, when the liquid medium is an aqueous medium, the substrate 1 is made of glass, metal, silicon wafer, or glass, metal, silicon wafer, resin, resin film, or the like whose surface is hydrophilized. Alternatively, the matrix layer 2 can be formed from a resin material capable of forming a hydrophobic surface, such as a glass, metal, silicon wafer, resin, or resin film having a hydrophilic surface formed by coating a hydrophilic layer.

On the other hand, when the liquid medium is non-aqueous, for example, the substrate 1 may be formed of a resin capable of forming a lipophilic surface, and the matrix pattern 2 may be formed of metal, glass, or the like capable of forming a hydrophilic surface. it can.

The substrate used for optically detecting the reaction is preferably an optically transparent substrate, and in some cases, an optically black substrate. As such a desirable substrate, a glass substrate such as synthetic quartz or fused quartz, a silicon wafer, a resin substrate such as acryl, polycarbonate, polystyrene, or vinyl chloride, or a substrate in which a black pigment or dye is mixed is used. Available. As the black pigment, carbon black or an organic black pigment can be used.

When the matrix pattern 2 is patterned by a fine processing technique, the resin forming the matrix pattern is a photosensitive resin, and the photosensitive resin is exposed and developed to easily form the matrix pattern. Can be formed.

In the present invention, the first region and the second region
It is desirable that the difference in affinity for the liquid medium with the region is larger. For example, when the liquid medium is aqueous, the substrate surface has higher hydrophilicity and the surface of the matrix pattern convex portion has higher hydrophobicity. It is desirable to have the property. In this case, when the liquid medium is an aqueous medium, the hydrophobicity of the matrix pattern can be enhanced by baking the exposed and developed matrix pattern, so that this method is one of the desirable embodiments of the present invention. I can say. Similarly, using the formed matrix pattern as a mask,
The hydrophilicity of the first region can be enhanced by dry-etching the substrate surface exposed in a pattern.

The constituent material of the matrix pattern used in the present invention may be any material that satisfies the requirements of the present invention. For example, a metal such as chromium, aluminum, and gold may be used. In particular, black chrome has a high light-shielding property, and is therefore desirable from the viewpoint of reliability when optical detection is performed in combination with a transparent substrate. However, in the case of metal, the hydrophilicity is rather high,
When a film is formed by means such as vapor deposition in consideration of the uniformity of the film thickness or the like, a film thickness of several thousand angstroms is generally used.

When the reaction system solution is aqueous, the first region is hydrophilic, so that the matrix pattern material is relatively hydrophobic and 1 μm in comparison with the substrate.
A material that can secure a film thickness of about m or more is preferable. For example, resin materials such as acrylic, polycarbonate, polystyrene, polyethylene, polyimide, acrylic acid monomer, urethane acrylate, and Teflon can be mentioned as preferable materials.

In this case, as one method of forming a matrix pattern, there is a method in which a photoresist is coated on a resin coated on the substrate surface, and after patterning, the resin is patterned by a process such as etching. In addition, if it is a photosensitive resin, the resin itself can be patterned by a photolithography process using a photomask. As such a photosensitive resin, 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 bisazide-based resist and 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,
Radiation-decomposable polymer resists such as polymethylene sulfone, polyhexafluorobutyl methacrylate, polymethyl isopropenyl ketone, and poly 1-trimethylsilylpropyne bromide, and dissolution inhibitor-based resists such as cholic acid-0-nitrobenzyl ester And polyvinyl phenol- as a negative resist.
3,3′-diazidodiphenyl sulfone, polyglycidyl methacrylate and the like can be mentioned.

As the ultraviolet curing resin, one kind selected from benzophenone and its substituted derivatives, benzoin and its substituted derivatives, acetophenone and its substituted derivatives, and oxime compounds such as benzyl oxime, etc. Alternatively, polyester acrylates, epoxy acrylates, urethane acrylates, etc. containing about 2 to 10% by weight of two or more photopolymerization initiators can be given.

As described above, the material for forming the matrix pattern is desirably a light-shielding material in consideration of improvement in sensitivity and reliability of optical detection, for example, when a fluorescence method is used. Examples of such a material include a metal, a black resin, and a black photosensitive resin as described above. Examples of the black resin or the black photosensitive resin include the above-described resins or those obtained by adding a black dye or a black pigment to the photosensitive resin. As a black pigment,
Carbon black and black organic pigments can be used. Such a black matrix pattern is called a black matrix.

The shape of the first region can be selected in consideration of easiness of formation, handling, and operability in detection when performing detection. Although not limited, etc., a simple shape as shown in FIG. 1 is desirable. The arrangement of the first regions may be arranged at equal intervals in the vertical direction in the plan view as shown in FIG. 1, or the first regions may be arranged so as to be shifted in adjacent rows, as desired. It can be changed as appropriate.

The size of each first region defined by the matrix pattern is 300 μm when considering the number of reactive species and the size of the entire array.
The following is desirable. For example, as shown in FIG. 1, when the cross-sectional shape formed together with the concave matrix pattern is a square, the length of one side can be 200 μm or less. Further, when the cross-sectional shape is rectangular, the long side is more preferably 200 μm or less, and when it is circular, the diameter is more preferably 200 μm or less. The lower limit of the size can be, for example, about 1 μm.

The distance between adjacent first regions is determined by considering the size of the entire array, cross contamination, operability in supplying various solutions, and the like. , 1/2 of the longest width of the first area
It is desirably in the range of up to twice. The thickness of the matrix pattern (height from the substrate surface) is determined in consideration of the method of forming the matrix pattern, the capacity of the first region, the amount of the reaction solution to be supplied, and the like. 20 μm
It is desirable that: The lower limit can be about 1 μm.

By setting this range, a considerable number of reaction fields are provided, and the application of the liquid medium to the first region during the reaction of two or more kinds of substances in the combinatorial chemistry is performed using an ink jet recording method. When this is performed, the order is picoliter to nanoliter as described later, but the discharge amount is not always constant due to fluctuations in discharge conditions and the like.
Even in such a case, by causing the first regions to be separated from each other by a convex matrix pattern having a height of 1 μm or more, the occurrence of cross contamination in the adjacent first regions can be suppressed.

The first region having a square planar shape is 200 μm.
When formed in a size of m × 200 μm × 20 μm, the volume is 800 pl. In addition, in the configuration of FIG.
If the distance x between the regions is 200 μm, 625 / c
A small reaction field density of m ² results. That is, an array having an order of 10 ² / cm ² or more can be included in the scope of the present invention. The first region is formed in a size of 5 μm × 5 μm × 4 μm, and the distance between adjacent first regions is also 5 μm.
m, the volume of each first region is 0.1 pl,
The density of the reaction field is 1,000,000 pieces / cm ² . Since the patterning of 5 μm × 5 μm × 4 μm is a realistic size in the current fine processing technology, an array of 10 ⁶ / cm ² or more can be included in the scope of the present invention.

Next, the reaction using the above-described reaction field array will be described.

In the present invention, at least predetermined portions of the plurality of first regions of the reaction field array having the above-described structure can react at least two kinds of substances in a liquid medium. In this case, the same reaction is performed between the first regions, or different reactions (including a case where the reactive substances constituting the reaction system are different or a case where the reactive substances are the same but the concentrations are different) are performed in parallel. be able to.

The formation of a reaction system in the first region can be carried out by a commonly used method. For example, when two kinds of substances are reacted, one of them is placed in a predetermined first region. A method can be used in which a solution containing the substance is first supplied, and then a solution containing the other substance is mixed with the solution to start the reaction. Further, in the case of three types, a method of adding one by one into the predetermined first region and mixing them, and a method of mixing the solution containing two of these and the solution containing the remaining one in the predetermined first region For example, a method of supplying the mixture to the inside and mixing can be used.

At this time, when the first region is formed as a minute region, for example, the amount of the reaction system solution is changed from sub-pl to sub-n.
When the amount is 1 and a small amount, it is desirable to use a method for preventing evaporation and air diffusion of the solution supplied under the reaction conditions. For example, when the reaction solution is an aqueous solution, it can be said that it is desirable that the array is placed in a constant temperature and constant humidity condition that requires conditions.

In the case of the above-mentioned example, when the volume of the solution supplied during the reaction in the present invention is set to be approximately the same as the volume of the first region, from the above calculation, it is approximately 0.1 pl to 1
nl. Further, in the present invention, since the matrix pattern is incompatible with the solution to be supplied, depending on the type of the liquid, an amount of the liquid exceeding the volume of the concave portion rises above the concave portion opening due to the surface tension and remains there. Becomes possible. In such a case, for example, a liquid amount 10 to several tens times the concave volume can be supplied. That is, in the case of the above example, several pl to several tens nl of liquid are supplied. In any case, it is often difficult to satisfactorily supply such a small amount of liquid with a general microdispenser or micropipette with good positional accuracy and supply amount accuracy. Therefore, in the present invention, it is preferable to supply the reaction solution to the concave portion by using the ink jet method in the present invention.

For supplying the reaction system solution by the ink jet method, for example, an ink jet head used in an ink jet printer can be used. In ink-jet printing, ink is ejected after positioning with high precision in the order of μm, and therefore, it can be said that it is extremely suitable for supplying a reaction system solution to the small reaction area array of the present invention. The amount of ink to be ejected is generally about 1 pl.
To several nl, it can be said that it is similarly suitable for supplying the reaction solution of the present invention. Since the ink jet head is manufactured using a semiconductor manufacturing technique, it is possible to further adjust the ejection amount to a desired amount.

The DN using such an ink-jet method
As described above, the sequential synthesis method of the A probe array is described in International Publication WO 95/25116.
The substrate described in this publication is simple glass or porous glass. In the case of simple glass, the area where the liquid spreads cannot be controlled, and cross contamination is also a problem. In the case of porous glass, the spread of droplets can be controlled to some extent but is not quantitative, and the problem of cross-contamination remains. Furthermore,
In the case of the ink jet method, there is a problem that the direction of the droplet is disturbed to a certain extent when the droplet is ejected from the head. However, in the case of simple glass or porous glass, the problem is that the disturbance is directly arranged in the array. There is also. On the other hand, according to the method of the present invention, the spread of the liquid is quantitatively controlled by the convex matrix pattern, and even if the ejection direction is disturbed, the dispersion is within the first region or the first region.
Even if the area deviates to a certain extent, the desired first area can be supplied due to the incompatibility of the matrix pattern with the ejection liquid.

The ink jet method capable of supplying the reaction solution of the present invention includes a piezo jet method,
A bubble jet method utilizing thermal foaming can be used.

Next, reactive species suitable for the reaction using the reaction field array of the present invention will be described. In the present invention, anything that can react within the first region arranged in the array may be used. However, when the liquid medium of the reaction system solution is aqueous, the first region needs to be hydrophilic, the surface of the convex matrix pattern needs to be hydrophobic, and when the solvent is an organic solvent, Requires that the first region be lipophilic and the surface of the convex matrix pattern be non-lipophilic. When two or more solutions are supplied to one of the first regions, it is desirable that the solvents forming the respective solutions are compatible.

Examples of the reactive species used in the present invention include ligands and receptors for the ligands, oligos having a specific amino acid sequence, polypeptides and substances having an affinity for these peptides, enzymes and substrates for the enzymes. An antigen and an antibody to the antigen, a nucleic acid having a specific base sequence, or a modified nucleic acid, the nucleic acid, or a nucleic acid having a base sequence complementary to a specific base sequence of the modified nucleic acid, or a modified nucleic acid. I can give it.
Examples of these nucleic acids and modified nucleic acids include DNA, RNA, or PNA whose backbone is composed of a peptide.
(Protein nucleic acid) and the like.

In the present invention, a form in which at least one kind of the substance to be reacted is bonded in the concave portion is also one of the forms of the present invention. Examples of the form of such a reactive species include:
Examples include an immobilized enzyme, an immobilized antibody, an immobilized nucleic acid probe, and an immobilized peptide probe. If at least one of the reactive species is immobilized, there is an advantage that the subsequent supply of other reactive species is simplified and that washing and the like can be easily performed.

Hereinafter, a series of manufacturing steps of a reaction field array when a black photosensitive resin is used as a matrix pattern material using a transparent glass substrate, and an example of a reaction method in the array will be described. (1) A transparent glass substrate is appropriately washed and dried, and a black photosensitive resin is applied. Various methods such as spin coating, a method using a die coater, and dip coating can be used as the method of application. (2) Temporarily cure the applied layer using, for example, a hot plate. Thereafter, exposure is performed using an exposure apparatus having a wavelength matching the spectral sensitivity of the photosensitive resin composition and a photomask having a predetermined pattern. (3) If a negative photosensitive resin composition is obtained by performing development, a portion that is shielded by a mask during exposure is eluted with a developing solution, the substrate surface is exposed, and the exposed portion remains as a black matrix. Subsequently, rinsing is performed to wash away the developer, and drying is performed. (4) Re-curing with a hot plate or the like to impart necessary water repellency. (5) Dry etching is performed using the black matrix as a mask so that the concave portions in the matrix pattern have the required cleanliness. (6) A solution of a substance to be reacted (in this case, an aqueous solution) is supplied to a concave portion in a matrix pattern at a desired position by a bubble jet method. (7) The substrate is placed under predetermined reaction conditions. (8) Perform necessary detection operation.

[0057]

EXAMPLES The present invention will be specifically described below with reference to examples.

Example 1 [Preparation of micro reaction field array using black matrix (for aqueous reaction solution)] A synthetic quartz substrate was subjected to ultrasonic cleaning using a 2% aqueous sodium hydroxide solution, and then subjected to UV ozone treatment. DEEP- containing carbon black
A UV resist (Nippon Steel Chemical Co., Ltd., negative type resist for black matrix BK-739P) was applied by a spin coater so that the film thickness after curing became 5 μm. This substrate was heated and cured at 80 ° C. for 5 minutes on a hot plate.

Using a DEEP-UV exposure apparatus, the width of the black matrix pattern (distance between concave portions in FIG. 1:
x, the same applies hereinafter) is 200 μm, 200 μm × 200 μm
Proximity exposure using a predetermined pattern mask corresponding to the square concave portion of the above, then developing with a developing solution of an inorganic alkali aqueous solution using a spin developing machine to form a black matrix, and further rinsing with pure water After processing, the developing solution is completely removed, and then dried easily using a spin drier.
By heating for 0 minutes, the black matrix was fully cured.

Next, the substrate surface was cleaned by performing a plasma ashing process on the substrate surface using the black matrix as a mask. At this time, when the contact angle of the black matrix surface with water was measured, the contact angle with water was 87 °, which is hard to wet, and the contact angle with water on the substrate surface, which was 22 °, was easily wet.

Example 2 [Preparation of micro reaction field array using black chromium (for non-aqueous reaction solution)] An acrylic resin (Asahi Kasei Kogyo Delaglass) substrate was ultrasonically cleaned using a 2% aqueous sodium hydroxide solution. After the UV ozone treatment, a pattern corresponding to the same arrangement of the first region as in Example 1 was formed of a resist by a normal photolithography process. The resist was applied so that the thickness after curing became 1 μm, and post-baked at 100 ° C. for 30 minutes. Next, after forming a black chrome film to a thickness of 2000 Å on the substrate surface, using a resist stripper,
A portion corresponding to the first region of the chromium film was removed by a lift-off method. After appropriate washing and drying, the substrate surface was cleaned by plasma ashing as in Example 1. This allows the matrix pattern to be black chrome,
A small reaction field array in which the first region was formed of an acrylic resin was obtained. At this point, the contact angle of the black chromium surface with water was measured to be 25 °, indicating that the surface was easily wetted. The contact angle of the first region with water was 93 °.
° and it was hard to get wet.

Example 3 [Supply of Aqueous Solution to Micro-Reaction Field Array by Inkjet Method I] A 100 μm black matrix pattern width, 100 μm × 100 μm × 1 cm × 1 cm area was formed on a glass substrate in the same manner as in Example 1. A small reaction field array having 2500 square first regions of 100 μm was formed. Then, a 10 μM aqueous solution of rhodamine B was added
Each of the first regions was supplied with 50 pl of the checkerboard pattern (every other) in an amount equivalent to the volume of the first region by the ink jet device. The ejection positioning accuracy of the used ink jet device is ± 2.5 μm. Next, 50 pl of an aqueous solution of 10 μM amino FITC was supplied to another first region from another inkjet head. The use of rhodamine B and amino FITC is water-soluble,
It is also for observing the state of the liquid supplied by the fluorescence and the state of the cross contamination.

A G excitation filter (for rhodamine B) and a B excitation filter (amino FITC)
) Was attached, and the state of each supplied aqueous solution was observed with fluorescence at a magnification of 100 times. Each aqueous solution was uniformly supplied in the first region without forming droplets. Also,
No fluorescence of the other dye was observed from the first region to which each dye was to be supplied, and it was possible to supply an aqueous solution without cross-contamination to each first region.

Example 4 [Supply of Aqueous Solution to Micro-Reaction Field Array by Inkjet Method II] In exactly the same manner as in Example 3, rhodamine B and amino FITC aqueous solution were applied to each first region. 500 pl which is 10 times the volume of one area
Supplied. When observed using a fluorescence microscope,
In the area, the aqueous solution of each dye, which was blocked by the water-repellent matrix pattern, was supplied in the form of droplets. Cross contamination was not observed as in Example 3.

Example 5 [Supply of Non-Aqueous Solution to Micro-Reaction Field Array by Inkjet Method] A 100 μm black chrome pattern width, 100 μm × A small reaction field array having 2500 square first regions of 100 μm was formed. Then, a 10 μM solution of rhodamine B in DMSO was supplied by an ink jet device to each of the first regions in a checkered pattern (every other one) at 50 pl, which was 50 times the volume of one of the first regions. Next, a 10 μM FITC DMSO solution was supplied from another inkjet head to the remaining first region. Observation using a fluorescence microscope showed that each first region had a raised DMSO solution of each dye, which was blocked by the non-lipophilic matrix pattern (the supply amount was not large enough to form a liquid solution. ) Had been supplied. Cross contamination was not observed as in Example 3.

Example 6 [Model of Supply of Two Aqueous Solutions to Micro-Reaction Field Array by Ink-Jet Method] In exactly the same manner as in Example 3, the first reactive species was added to each first region. As a model, an aqueous solution of rhodamine B and amino FITC was supplied at 250 pl, which is 5 times the volume of one volume in the first region.
Next, as a model of the second reactive species, rhodamine B and an amino FITC aqueous solution were supplied to the same position at 250 pl, which is 5 times the volume of one volume in the first region. When observed using a fluorescence microscope, each first region was blocked by a water-repellent matrix pattern,
In addition, the aqueous solution of each dye was supplied in an appropriate form without any splash of the liquid due to the second supply of the liquid. Also, no cross contamination was observed.

Example 7 [Supply and Reaction of Two Aqueous Solutions to Micro-Reaction Field Array by Inkjet Method] In the same manner as in Example 3, each of the first regions was subjected to sonication to fragment salmon tests. 4 as DNA base pairs
50 pl of a 0 μM TE (pH 7.5) buffer solution was supplied. Next, 10 μM of ethidium bromide (EB) was used.
Of TE (same as above) buffer solution was supplied at 25 ° C.
After 5 minutes in a thermo-hygrostat at 100% humidity, the fluorescence was observed with a fluorescence microscope (G excitation filter). As a result, EB-derived fluorescence was observed from each first region. This is because the EB reacts with the double-stranded nucleic acid in the first region,
This shows that the fluorescence from the intercalated EB was observed. Only weak fluorescence was observed from the first region to which only EB was supplied as a control.

Example 8 [Supply and Reaction of Two Aqueous Solutions to Micro-Reaction Field Array by Inkjet Method, and Quantification of Fluorescence After Reaction I] Eight primary liquids were obtained in the same manner as in Example 7. In the region of 0 μM, 2 μM, 5 μM, 10 μM,
20 μM, 40 μM, 100 μM and 200 μM DN
A solution of 50 pl was supplied. Then, 50 μl of a 10 μM EB solution was supplied to each of the first regions, and Example 7 was performed.
After placing in a constant temperature and humidity chamber as in
Images are captured by a CD camera (Hamamatsu Photonics C2400-87) and processed by an image processing device (Hamamatsu Photonics Arg)
us50). Argus
The signal amplification levels of 50 were appropriately set. The results are shown in FIG. From FIG. 2, it can be seen that the reaction in the small reaction field array of the present invention proceeded and was detected with good quantitativeness. The reason why the fluorescence intensity reaches the saturation state is due to the quantitative ratio of DNA and EB.

Example 9 [Supply and Reaction of Two Aqueous Solutions to Small Reaction Field Array by Inkjet Method, and Quantification of Fluorescence After Reaction I
I] Carboxyfluorescein diacetate, a fluorescent substrate of esterase (Molecular Probes, C
-369) in a 40 μM TE buffer solution (pH 7.5)
Were respectively applied to seven first regions of a micro-reaction field array prepared in the same manner as in Example 3 by an ink-jet method.
pl. Then, a reaction solution of 2 units / 1 cholinesterase (Wako Pure Chemical Industries, Ltd.) in TE buffer (same as above) was added for 0 minutes, 5 minutes, 10 minutes, 15 minutes, and 20 minutes.
, 25 minutes and 30 minutes. During that time, the reaction was at a temperature of 25 ° C and a humidity of 100%
In a constant temperature and humidity chamber. After that, the mixture was treated at 60 ° C. for 5 minutes to dissipate the enzyme, and then the fluorescence from carboxyfluorescein generated by the enzyme digestion was observed with a fluorescence microscope (B excitation filter) + ICCD + Argus.
Quantified. The signal amplification level of Argus 50 is the same as that of the eighth embodiment. The results are shown in FIG. FIG. 3 shows that the enzymatic reaction in the micro-reaction field array of the present invention proceeded and was detected with good quantitativeness. The reason why the fluorescence intensity reaches the saturation state is due to the quantitative ratio between the enzyme and the substrate.

Example 10 [Fabrication of further fine reaction field array and supply of aqueous solution by ink-jet method] In the same manner as in Example 1, an area of about 1 cm × 1 cm on a glass substrate was
5 μm black matrix pattern width (film thickness 2 μm),
A first area of 5 μm × 5 μm square is defined as 1,000,000
A micro-reaction field array having 00 pieces was formed. Next, as in Example 3, 1 pl of a 10 μM aqueous solution of rhodamine B was supplied to each first region in a checkered pattern using an inkjet device. The ejection positioning accuracy of the used ink jet device is ± 0.5 μm. Next, 1 pl of an aqueous solution of 10 μM amino FITC was supplied to the remaining first region from another inkjet head.

G excitation filter (for Rhodamine B) and B excitation filter (Amino FITC)
) Was attached, and the state of each supplied aqueous solution was observed by fluorescence at 400 ×. In each of the aqueous solutions, the aqueous solution of each dye was supplied in the form of a liquid as in Example 4. Also, no cross contamination was observed.

[0072]

According to the present invention, it has become possible to produce a micro-reaction field array suitable for supplying a reactant solution for performing a large number of kinds of reactions on a substrate, for example, in an area of 1 cm × 1 cm. . In addition, the combined use of the ink jet method enables the reaction such as the supply of the reaction species solution to the micro reaction field array of the present invention, and the detection and quantification of the reaction as required.

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of the structure of a reaction field array according to the present invention, wherein (A) is a plan view and (B) is a longitudinal sectional view.

FIG. 2 is a diagram showing the results of fluorescence quantification of Example 8.

FIG. 3 is a diagram showing the results of fluorescence quantification of Example 9.

[Explanation of symbols]

 Reference Signs List 1 substrate 2 matrix pattern 3 first region 4 substrate surface

Claims (79)

[Claims] A plurality of reaction fields for reacting two or more kinds of substances in a liquid medium are constituted by a first region showing a predetermined affinity for the liquid medium, A method for producing a reaction field array wherein the reaction fields are separated from each other by a second region having a lower affinity for the liquid medium as compared to the affinity of the first region, wherein the liquid medium On the substrate surface showing a predetermined affinity for, the substrate surface has a lower affinity compared to the predetermined affinity shown for the liquid medium, a matrix pattern convex to the substrate surface Forming the substrate surface exposed corresponding to the matrix pattern as a first region;
A method for producing a reaction field array, wherein the matrix pattern is used as a second region. 2. The method according to claim 1, wherein the surface of the substrate is hydrophilic and the surface of the matrix pattern is hydrophobic. 3. The method according to claim 1, wherein the surface of the substrate is lipophilic, and the surface of the matrix pattern is non-lipophilic. 4. The method according to claim 1, wherein the matrix pattern is formed of a resin material. 5. The method according to claim 1, wherein the matrix pattern is formed using a photolithography method. 6. The photolithography method comprises forming a resin layer on the substrate surface, forming a photoresist layer on the resin layer, and exposing the photoresist layer in a pattern corresponding to the matrix pattern. Developing and forming a photoresist pattern on the resin layer; and patterning the resin layer using the photoresist pattern as a mask, and then removing the photoresist pattern to form the pattern. The method for producing a reaction field array according to claim 5, comprising: 7. The photolithography method comprises forming a photosensitive resin on the surface of the substrate, exposing the photosensitive resin layer to a pattern corresponding to the matrix pattern, and developing the pattern to form the pattern. The method for producing a reaction field array according to claim 5, comprising a step. 8. The method according to claim 7, wherein the pattern formed by patterning the photosensitive resin layer is further post-baked to improve water repellency. 9. The method according to claim 1, wherein the surface of the substrate is hydrophilic.
3. The method for producing a reaction field array according to claim 2, wherein the surface of the substrate constituting the region is etched using the second region as a mask to enhance the hydrophilicity of the first region. 10. The method according to claim 1, wherein the substrate is optically transparent, and the second region is light-shielding. 11. The method according to claim 1, wherein the second area is black.
0. The method for producing a reaction field array according to 0. 12. The method according to claim 1, wherein the height of the second region is 1 to 20 μm. 13. The method according to claim 1, wherein the distance between the first regions adjacent to each other is 1/2 to 2 times the longest width of the first regions. 14. A reaction field array having a plurality of reaction fields for reacting two or more kinds of substances in a liquid medium, wherein each reaction field is arranged separately from each other, wherein said reaction field is A first region having a predetermined affinity for the liquid medium, and the reaction field having a lower affinity for the liquid medium as compared with the affinity of the first region. A reaction field array isolated from each other by a second region that is convex with respect to the first region. 15. The reaction field array according to claim 14, wherein the density of the reaction field is not less than 10 ² / cm ² . 16. The reaction field array according to claim 14, wherein the second region is formed in a pattern on the surface of a substrate having a surface having a predetermined affinity for the liquid medium. 17. The reaction field array according to claim 14, wherein the second region is made of a resin material. 18. The reaction field array according to claim 16, wherein the second region is formed by a photolithography method. 19. The first area is optically transparent,
15. The reaction field array according to claim 14, wherein the second region is light-blocking. 20. The method according to claim 20, wherein the substrate is optically transparent,
15. The reaction field array according to claim 14, wherein the region is light-shielding. 21. The second area is black.
Reaction field array as described. 22. The reaction field array according to claim 14, wherein the longest width of the first region is 200 μm or less. 23. The distance between adjacent ones of the first regions, separated by the second regions, is in the range of 1/2 to 2 times the longest width of the first regions. 1
4. The reaction field array according to 4. 24. The reaction field array according to claim 14, wherein the height of the second region is 1 to 20 μm. 25. The method according to claim 25, wherein a plurality of reaction fields for reacting two or more substances in a liquid medium are supplied to each reaction field of a reaction field array arranged separately from each other. Wherein the reaction field array comprises a first region in which the reaction field exhibits a predetermined affinity for the liquid medium, and wherein the reaction field is A method of reacting a substance, wherein the substance has a lower affinity than the first region and is isolated from each other by a second region that is convex with respect to the first region. 26. The reaction method according to claim 25, wherein the density of the reaction field is 10 ² / cm ² or more. 27. The reaction method according to claim 25, wherein the second region is formed in a pattern on a surface of the substrate having a predetermined affinity for the liquid medium. 28. The reaction method according to claim 25, wherein the second region is made of a resin material. 29. The method according to claim 25, wherein the second region is formed by a photolithography method.
The reaction method described in 1. 30. The first region is optically transparent,
The reaction method according to claim 25, wherein the second region is light-shielding. 31. The method according to claim 31, wherein the substrate is optically transparent,
The reaction method according to claim 27, wherein the region is light-shielding. 32. The second area is black.
The reaction method described in 1. 33. The reaction method according to claim 25, wherein the longest width of the first region is 200 μm or less. 34. The reaction method according to claim 25, wherein a distance between the first regions adjacent to each other is in a range of 1/2 to 2 times a longest width of the first regions. 35. The reaction method according to claim 25, wherein the height of the second region is 1 to 20 μm. 36. The two or more substances include those having a ligand and receptor relationship with each other.
The reaction method described. 37. The reaction method according to claim 25, wherein the two or more kinds of substances include an oligopeptide or a polypeptide having a specific amino acid sequence. 38. The reaction method according to claim 25, wherein the two or more substances include a protein. 39. The reaction method according to claim 38, wherein the protein is an antibody. 40. The reaction method according to claim 38, wherein the protein is an antigen. 41. The reaction method according to claim 25, wherein the two or more substances include an enzyme. 42. The reaction method according to claim 25, wherein the two or more substances include a single-stranded nucleic acid having a predetermined base sequence or a modified nucleic acid. 43. The reaction method according to claim 42, wherein the nucleic acid is an oligonucleotide or a polynucleotide. 44. The reaction method according to claim 42, wherein the nucleic acid is DNA. 45. The reaction method according to claim 42, wherein the nucleic acid is RNA. 46. The modified nucleic acid is a protein nucleic acid PNA.
43. The reaction method according to claim 42, which is (Protein Nucleic Acid). 47. The reaction method according to claim 25, wherein the supply of the substance to the reaction field is performed using an inkjet method. 48. The reaction method according to claim 47, wherein the supply of the substance to the reaction field is performed by a bubble jet method. 49. The reaction method according to claim 47, wherein the supply of the substance to the reaction field is performed by a piezo jet method. 50. A method for quantifying a first substance contained in a sample solution, comprising: (a) a plurality of reactions comprising a first region exhibiting a predetermined affinity for the sample solution; Providing a reaction field array, wherein the reaction fields are separated from each other by a convex second region having a lower affinity for the sample solution as compared to the first region. (B) supplying the sample solution to each reaction field of the reaction field array; (c) a signal capable of quantitatively detecting the first substance by interaction with the first substance. Supplying a reagent providing the following to each reaction field of the reaction field array; and (d) quantitatively detecting an interaction between the first substance and the reagent. How to determine the substance. 51. The method according to claim 51, wherein the step (c) comprises the steps of: adding a liquid medium exhibiting a predetermined affinity of the first region, comprising a second substance capable of binding to the first substance and the reagent; 52. The method according to claim 50, further comprising the step of supplying the reagent to the reagent, wherein the reagent interacts with a conjugate of the first substance and the second substance. 52. The second substance supplied to the reaction field,
52. The method according to claim 51, wherein the method is different depending on each reaction field. 53. The method according to claim 50, wherein the density of the reaction field is at least 10 ² / cm ² . 54. The method according to claim 50, wherein the second region is formed in a pattern on the surface of a substrate having a surface having a predetermined affinity for the liquid medium. 55. The method according to claim 50, wherein the second region is formed of a resin material. 56. The quantification method according to claim 50, 54 or 55, wherein said second region is formed by a photolithography method. 57. The first area is optically transparent,
The method according to claim 50, wherein the second region is light-shielding. 58. The substrate, wherein the substrate is optically transparent and the second
The quantitative method according to claim 50, wherein the region is light-shielding. 59. The second area is black.
Or the quantification method according to 58. 60. The method according to claim 50, wherein the longest width of the first region is 200 μm or less. 61. A distance between the adjacent first regions separated by the second region is in a range of 1/2 to 2 times a longest width of the first region. 50. The quantification method according to 50. 62. The quantification method according to claim 50, wherein the height of the second region is 20 μm or less. 63. The method according to claim 50, wherein said reagent is contained in said sample solution. 64. The method according to claim 51, wherein the reagent is contained in a liquid medium containing the second substance. 65. The method according to claim 51, wherein the first and second substances have a relationship between a ligand and a receptor. 66. The method according to claim 51, wherein at least one of the first substance and the second substance is an oligopeptide or a polypeptide having a specific amino acid sequence. 67. The method according to claim 66, wherein at least one of the first substance and the second substance is a protein. 68. The method according to claim 67, wherein said protein is an antibody. 69. The method according to claim 67, wherein said protein is an antigen. 70. The method according to claim 51, wherein at least one of said first substance and said second substance is an enzyme. 71. The method according to claim 51, wherein at least one of the first substance and the second substance contains a single-stranded nucleic acid having a predetermined base sequence or a modified nucleic acid. 72. The method according to claim 71, wherein the nucleic acid is an oligonucleotide or a polynucleotide. 73. The method according to claim 71, wherein the nucleic acid is DNA. 74. The method according to claim 71, wherein the nucleic acid is RNA. 75. The modified nucleic acid is a protein nucleic acid (PN
A. The method for quantification according to claim 71, which is A: Protein Nucleic Acid). 76. The method according to claim 50, wherein at least one of the supply of the sample and the supply of the reagent to the reaction field is performed by an inkjet method. 77. The method according to claim 51, wherein the supply of the liquid medium containing the second substance to the reaction field is performed by an ink jet method. 78. The method according to claim 76, wherein the ink jet method is a bubble jet method. 79. The method according to claim 76, wherein the ink jet method is a piezo jet method.