JP2003014760A - Probe carrier, probe fixing carrier, and their manufacturing methods - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately and quickly manufacture a probe carrier that is improved in reliability (particularly, is made free from mixed liquids and voids) and increased in density and to accurately and quickly detect the position of a target compound specifically coupled with a probe fixed on the probe carrier. SOLUTION: A solution containing the probe is imparted to a specific position on the probe carrier having an index in a probe non-fixing area on the basis of the position of the index and the probe is fixed. Preferably, partition walls are formed on the carrier and the spreadability of the probe solution in the areas surrounded by the partition walls is improved by improving the water repellency of the partition walls by irradiating the walls with a plasma in a gas atmosphere containing fluorine atoms and then bringing water into contact with the carrier. In addition, the position of the target compound specifically coupled with the probe fixed on the probe carrier manufactured by the manufacturing method can be detected accurately and quickly on the basis of the index.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention particularly relates to a method for producing a probe carrier in which a probe solution is applied to a specific position on a carrier by using an index by an inkjet method, and a probe carrier produced by the method, Relates to a method for identifying the position of a target substance bound to a probe on the probe carrier by using the above index.

[0002]

2. Description of the Related Art When analyzing the base sequence of a gene DNA, or simultaneously performing highly reliable gene diagnosis on many items, a DNA having a target base sequence is selected using plural kinds of probes. Is required. DNA microchips have been attracting attention as a means for providing a plurality of types of probes used for this sorting operation. Also,
Even in high-throughput screening of drugs and combinatorial chemistry, many solutions of target proteins and drugs (eg, 96, 384, 1
It is necessary to arrange (536 kinds) and perform an orderly screening. Controls a method for arranging multiple kinds of drugs for that purpose, an automated screening technique in that state, a dedicated device, a series of screening operations,
Also, software and the like for statistically processing the results have been developed.

These parallel screening operations basically consist of a large number of known probes as a means for selecting substances to be evaluated, so-called probe.
By using an array, it is possible to detect the presence or absence of an action or reaction on the probe under the same conditions. In general, what kind of action and reaction to use for the probe is determined in advance. Therefore, the probe species mounted in one probe array may be, for example, a group of DNA probes having different base sequences. It is one type of substance when roughly classified. That is, the substance used for the group of probes is, for example, DNA, protein, or a synthesized chemical substance (drug). In many cases, a probe array composed of a plurality of types of probes is often used. However, depending on the nature of the screening work, as probes, DNA having the same base sequence, protein having the same amino acid sequence, and the same chemical It is also possible to use an array form in which a large number of substances are arranged. These are mainly used for drug screening and the like.

In a probe array consisting of a plurality of types of probes, specifically, a group of DNAs having different base sequences, a group of proteins having different amino acid sequences, or a group of different chemical substances is used. In many cases, a plurality of types of constituents are arranged in an array on a carrier according to a predetermined arrangement order.
Among them, the DNA probe array is used for analysis of the base sequence of gene DNA and, at the same time, for highly reliable gene diagnosis of many items.

One of the problems in a probe array consisting of a plurality of types of probes forming this group is to use as many types of probes as possible, for example, D having various types of base sequences.
The NA probe is placed on one carrier. In other words, how densely the probes can be arranged in an array.

[0006] As one method of immobilizing plural kinds of probes in an array on a carrier, US Pat. No. 5,424,424 has been used.
No. 186, a method for producing DNA probes having different base sequences in an array form by sequential extension reaction of DNA on a carrier using a photodegradable protecting group and photolithography. You can Using this method, for example, 1000 per 1 cm2
It is also possible to prepare a DNA probe array carrying zero or more kinds of DNAs having different sequences. In this method, when synthesizing DNA by a sequential extension reaction, a photolithography process is performed for each of four types of bases (A, T, C, G) by using a dedicated photomask, and a predetermined array is prepared. By selectively extending one of the bases at a position, a plurality of types of DNA having a desired base sequence are synthesized on the carrier with a predetermined sequence. Therefore, the longer the chain length of DNA, the higher the cost required for the preparation and the longer time required. In addition, since the efficiency of nucleotide synthesis in each extension step is not 100%, the ratio of DNA in which the designed nucleotide sequence has a defect is not small. Furthermore, in the case of using a photodegradable protective group in the synthesis, the synthesis efficiency is lower than in the case of using an ordinary acid-decomposable protective group,
In the finally obtained array, there is also a problem that the proportion of DNA according to the designed nucleotide sequence becomes small.

Further, since the product directly synthesized on the carrier is used as it is, it is, of course, impossible to remove the DNA having the defective base sequence from the designed DNA according to the designed base sequence by purification fractionation. . Other,
In the finally obtained array, there is a problem that the base sequence of DNA synthesized on the carrier cannot be confirmed. This is because if a given base was hardly extended at a certain extension stage due to an error in the process, and it was a completely defective product, the screening using this defective product probe array resulted in an incorrect result. However, it means that there is no way to prevent it. The inability to confirm this nucleotide sequence is the biggest and essential problem in this method.

As a method different from the above-mentioned method, DNA for a probe is previously synthesized and purified, and in some cases, after confirming the base length thereof, each DNA is applied onto a carrier by a device such as a microdispenser. And probe
Techniques for preparing arrays have also been proposed. PCT publication WO95 / 355O5 describes a method of applying DNA onto a membrane using a capillary. By applying this method, it is possible in principle to prepare about 1000 DNA arrays per cm 2. Basically, one capillary-type dispensing device for each probe is used to apply the probe solution to a predetermined position on the carrier, and this operation is repeated,
This is a method of preparing an array. There is no problem if you prepare a dedicated capillary for each probe, but if you try to do the same work with a small number of capillaries, to prevent cross-contamination, the capillaries should be sufficient when replacing the probe type. Need to be washed. Further, it is necessary to control the applied position each time. Therefore, it cannot be said to be a method suitable for preparing an array in which a large number of types of probes are arranged at high density. In addition, since the probe solution is applied to the carrier by tapping the tip of the capillary onto the carrier, reproducibility and reliability cannot be said to be perfect.

Another method is to use DN on a carrier.
When performing solid phase synthesis of A, a method has also been proposed in which a solution of a substance required for synthesis is applied onto a carrier by an inkjet method at each extension step. For example, European Patent Publication EP
No. 0 703 825 B1 discloses nucleotide monomers utilized in solid phase synthesis of DNA, and
A method for solid phase synthesis of a plurality of DNA species each having a predetermined nucleotide sequence by applying activators from different piezo jet nozzles is described. The application (application) by the ink jet method is more reliable than the application (application) of the solution using the capillary, such as the reproducibility of the applied amount, and the structure of the nozzle can be miniaturized. It has characteristics suitable for increasing the density of probe arrays. However, since this method also basically applies the sequential elongation reaction of DNA on a carrier, the above-mentioned US Pat.
The biggest problem in the method described in Japanese Patent No. 424,186, that is, the problem that the base sequence of DNA synthesized on the carrier cannot be confirmed still remains. Although the complexity of performing a photolithography process using a dedicated mask for each extension step is eliminated, the point that a predetermined probe is fixed at each point, which is an essential requirement for the probe array. , With some problems. The EP
No. 0,703,825 B1 describes only a method of using a plurality of piezo jet nozzles formed independently, and when using this small number of nozzles, a method using the above-mentioned capillary is used. Similarly, it is not always suitable for high density probe array preparation.

Further, Japanese Patent Application Laid-Open No. 11-187900 discloses a method in which a liquid containing a probe is attached as droplets to a solid phase by a thermal ink jet head to form a spot containing the probe on the solid phase. There is.

A probe using an inkjet method
In array preparation, it is desirable to immobilize as many kinds of probes as possible within a certain range in order to improve the detection efficiency in diagnosis and the like from the viewpoint of high density, and not to prepare a large amount of sample. Further, from the viewpoint of high reliability, a specific probe needs to be provided at a desired position. In order to prevent an error at the time of diagnosis or the like, it is desirable that only a desired probe is fixed at a predetermined place on the probe carrier.

[0012]

As described above, in order to fix the probes with higher density in the future, further position control will be required. However, in the conventional method of fixing the probe using the inkjet method, the position of the carrier and the head is adjusted while visually confirming the entire carrier to eject the liquid, so that the liquid can be applied to a desired position. There were times when I couldn't.

Further, when the probe carrier is manufactured by the ink jet method divided into a plurality of conventional times, the carrier and the head may be aligned each time. In this case, the method of discharging again after confirming the entire position of the applied probe after actually discharging the probe carrier is adopted. However, when the probe solution applied on the carrier is dried first, it is not known at which position on the carrier the probe is applied. However, there is an inconvenience such as the necessity of discharging In addition, this method causes inconvenience such that alignment is time-consuming because it is performed by human eyes and it is not accurate because many measurement points cannot be obtained. Furthermore, when the probe carrier is set upside down with respect to the head during manufacturing of the probe carrier, it cannot be judged only from the carrier when the carrier is transparent, and this mistake is overlooked in the inspection process. There is a possibility that it will end up.

Further, in JP-A-11-099000, in the probe carrier, in order to enhance the contrast in detection by fluorescence or the like, a partition wall of a light shielding layer (called a black matrix) is formed on the carrier by using a photolithography method or the like. It is disclosed that by forming it, it is fixed at a desired position. However, according to the earnest research of the present inventors, if the head is not precisely aligned with the probe carrier, the probe solution may not be able to interact with the opening (well) of the adjacent partition when ejecting the probe by the inkjet method. It was found that the desired probe could not be applied to the desired position due to the mixture with the above (referred to as mixed solution), and the characteristics as the probe carrier might be impaired. In addition, if the ejection position shifts, the spread of the probe solution in the well becomes uneven and the surface of the carrier is exposed a lot, resulting in uneven detection, difficulty in quantification, white bright spots (referred to as “white spots”), and non-specification. It was found that further problems such as an increase in physical coupling also occur. The “white spot” is a defect that occurs because the discharged probe solution does not spread sufficiently in the region surrounded by the partition walls, which causes unevenness of the probe applied on the carrier. This unevenness is D
When hybridized with a sample such as NA, it not only binds to only the probe having a specific base sequence immobilized on the carrier, but also adheres to the substrate (eg, glass) exposed in the blank areas. Also occurs, which causes a decrease in fluorescence contrast during measurement. The conceptual diagram is shown in FIG. In FIG. 3, 31 is a transparent substrate, 32 is a partition wall, 33 is a probe solution, and 34 is a blank portion. Moreover, (b) is an AB sectional view in (a). As shown in FIG. 3, “white spots” means that the probe solution 33 is difficult to spread in a complicated corner portion or a portion where the opening width is narrow, and the thickness of the probe solution 33 is small near the partition wall 32. This is a problem that occurs because the color becomes whiter than other parts. White spots decrease as the amount of the probe solution applied increases, but do not completely disappear.

A photoresist is usually used for forming the partition 32, and various components contained in the photoresist may adhere and remain in the opening of the partition 32. This adhered residue may hinder the wetting and spreading of the probe solution. Therefore, there is a demand for improvement in order to obtain a probe carrier with higher reliability and higher definition, and to obtain a probe carrier with higher yield.

Furthermore, shortening the time required for alignment is an important issue in shortening the chip manufacturing time,
The position adjustment by visual observation and the method of performing alignment by confirming the overall shape of the black matrix by using a detector take a very long time, resulting in an increase in cost per chip.

The present invention has been made on the basis of the recognition of such a new technical problem, and the purpose thereof is to displace the ejection position of the probe solution or to dispose the probe solution when applying the probe solution in the probe fixing region. It is an object of the present invention to provide a probe carrier that solves problems such as liquid mixture and white spots between adjacent probe fixing regions caused by insufficient wetting and spreading.

More specifically, when the ink is ejected onto the carrier for immobilizing the probe by the inkjet method, the ejection head is accurately and quickly positioned with respect to the carrier, or the target substance is detected or quantified when the target substance is detected or quantified. It is an object of the present invention to provide a probe immobilizing carrier and a probe carrier that can be accurately and quickly positioned.

Further, in order to provide a highly reliable probe carrier with a high yield, when a probe solution is applied to the probe fixing region surrounded by the partition wall, a mixed solution between adjacent probe fixing regions is prevented, In addition, the probe solution is sufficiently diffused in the region to form a probe carrier without white spots.

[0020]

Therefore, in order to solve the above-mentioned problems, in the first embodiment of the present invention, a plurality of kinds of probes capable of specifically binding to a target substance on a carrier are provided at different specific positions. A method for producing a fixed probe carrier, which comprises, in the aspect of the present invention, providing an index on a portion of the carrier other than the specific position, and including the probe based on the position of the index. A method for producing a probe carrier, comprising: a step of applying a solution to the specific position on the carrier; and a step of fixing the probe on the carrier.

In the embodiment of the present invention, the carrier is
It is preferable that a partition for partitioning the specific position is provided, and the index is at a predetermined position of the partition.

In the above aspect of the present invention, a step of forming a partition wall for partitioning the specific position on the carrier, and plasma irradiation for irradiating the carrier with plasma under an atmosphere of a gas containing at least fluorine atoms. It is preferable to further include a step and a step of removing the gas component attached to the specific position.

In the aspect of the present invention, it is preferable that the gas component removing step is a water washing step of washing the gas component by bringing water into contact with the surface of the carrier irradiated with the plasma.

In the aspect of the present invention, it is preferable that the partition wall is formed by a photolithography method.

In the above aspect of the present invention, in the photolithography method, a photosensitive resin layer is formed on the surface of the carrier, the photosensitive resin layer is exposed in a pattern corresponding to the partition walls, and developed. It is preferable that the method includes a step of forming the partition wall, and further includes a step of firing the partition wall prior to the plasma irradiation step.

In the aspect of the present invention, it is preferable that, in the washing step, the carrier is brought into contact with water and then dried at 100 ° C. or lower.

In the aspect of the present invention, it is preferable that the index is provided at a predetermined position of the partition wall.

In the above aspect of the present invention, it is preferable that the step of applying the solution containing the probe to the specific position is performed by flying the solution in a space.

In a second aspect of the present invention, there is provided a method for producing a probe-immobilizing carrier for immobilizing a plurality of types of probes capable of specifically binding to a target substance on different specific positions on a carrier, A step of forming partition walls for partitioning a region on the carrier, a plasma irradiation step of plasma-irradiating the carrier under a gas atmosphere containing at least fluorine atoms, and water on the surface of the carrier subjected to the plasma irradiation. And a step of washing with water to wash the gas component in contact therewith.

In a third aspect of the present invention, a solution containing the probe is applied to the probe-immobilizing carrier produced by the above method at the specific position on the carrier to immobilize the probe. A method for manufacturing a probe carrier is provided.

In the fourth aspect of the present invention, there is provided a probe carrier in which plural kinds of probes capable of specifically binding to a target substance are immobilized at different specific positions, and the specific positions on the carrier are excluded. Provided is a probe carrier having an index on a portion thereof.

In the embodiment of the present invention, the carrier is
It is preferable that a partition for partitioning the specific position is provided, and the index is at a predetermined position of the partition.

In the above aspect of the present invention, the index is
It is preferable that the concave portion is provided in the partition wall.

In the embodiment of the present invention, the partition wall is
It is preferably light-shielding.

In the aspect of the present invention, it is preferable that the partition wall is formed of a resin composition containing carbon black.

In the above aspect of the invention, the partition wall is water repellent, and the partition wall has a contact angle of 120 with pure water.
It is preferable that the probe fixing region is hydrophilic, and the contact angle of the probe fixing region with respect to pure water is 30 ° or less.

In the above aspect of the present invention, the average length of the partition cross-section upper portion in the direction parallel to the substrate at a height between 80% to 100% of the height of the partition top from the bottom of the partition cross section. It is preferable that the relationship between the value a and the length b of the lower bottom in the cross section of the partition wall has a shape represented by the equation 1.3 ≧ a / b ≧ 0.7.

[0038] In the fifth aspect of the present invention, there is provided a probe-immobilizing carrier for immobilizing a plurality of types of probes capable of specifically binding to a target substance at different specific positions, the specific carrier being on the carrier. Provided is a carrier for probe immobilization, which has an index in a portion except a position.

In the embodiment of the present invention, the carrier is
It is preferable that a partition for partitioning the specific position is provided, and the index is at a predetermined position of the partition.

In the above aspect of the present invention, the index is
It is preferable that the concave portion is provided in the partition wall.

In the embodiment of the present invention, the partition wall is
It preferably has a light-shielding property.

In the embodiment of the present invention, the partition wall is
It is preferably formed of a resin composition containing carbon black.

In the above aspect of the invention, the partition wall is water repellent, and the partition wall has a contact angle of 120 with pure water.
It is preferable that the fixing area of the probe is hydrophilic and the contact angle of the fixing area of the probe with respect to pure water is 30 degrees or less.

In the above aspect of the present invention, the average length of the partition wall upper portion in the direction parallel to the substrate at a height between 80% and 100% of the height of the partition wall from the bottom of the partition wall. It is preferable that the relationship between the value a and the length b of the lower bottom in the cross section of the partition wall has a shape represented by the equation 1.3 ≧ a / b ≧ 0.7.

In the sixth aspect of the present invention, a probe specifically bound to the target substance by imparting the target substance to a plurality of types of probes capable of binding to the target substance, which are carried on a carrier, is provided. A method for specifying a position, wherein the position of the probe specifically bound to the target substance is specified based on an index provided at a position different from the specific position where the plurality of types of probes are carried. A specific method is provided.

In the above aspect of the present invention, it is preferable that the index and the target substance are labeled with a radioactive label.

In the above aspect of the present invention, it is preferable that the index and the target substance are labeled with a fluorescent label.

In the embodiment of the present invention, the carrier is
The carrier is a light-transmissive plate-like body, and has a partition wall for partitioning a specific position where the plurality of types of probes are immobilized on one surface of the carrier, and the radioactive label is emitted. It is preferable to detect the fluorescence penetrating the carrier on the other surface of the carrier.

The present invention is particularly directed to a method for producing a probe carrier in which a probe solution is applied to a specific position on a carrier by using an index by an inkjet method, a probe carrier manufactured by the method, and further the above index. The present invention relates to a method for identifying the position of a target substance bound to a probe on the probe carrier.

[0050]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below with reference to the drawings.

FIG. 1 is a view exemplifying one embodiment of the probe carrier or probe-immobilizing carrier of the present invention on which an index is formed. In FIG. 1, 11 is an index, 12 is a black matrix, 13 is an opening (well), 14 is each chip, and 15 is a chip peripheral part. In FIG. 1, the index 11 is surrounded by a black matrix 12. , Forming a concave opening. FIG. 1 is an example, and the indices used in the method for producing a probe carrier according to the present invention will be described in detail below.

The index used in the production of the probe carrier according to the present invention is typically used for alignment of position adjustment between the carrier and the probe applying means when the probe is fixed. In the present embodiment, the index is used as an alignment mark when fixing the probe in a so-called inkjet method in which a probe solution containing a probe is discharged onto a carrier from a discharge head, and the probe is discharged onto the probe carrier. The liquid ejection position can be accurately specified and the alignment mark can be adjusted quickly, but the present invention is not limited to this configuration. Any means may be used as long as it is a method that requires positional adjustment between the carrier and the probe applying means at the time of fixing. Furthermore, the index may be used as a point for alignment of a scan image at the time of detection or for obtaining address information of a plurality of probe positions at the time of detection. Further, the present invention has a remarkable effect on these combinations. For example, it is used as an alignment mark between the carrier and the applying means when the probe is fixed, then the index is used as an alignment for probe detection, and the address information of each probe is obtained from the index. The effect of being able to perform is also a feature of the present invention.

The index may be convex or concave with respect to the carrier, or may be formed in the carrier. For example,
A concave protrusion or the like can be provided on the carrier, and the protrusion can be formed into a desired shape by photolithography using a resin material or the like. When the carrier is a glass substrate or the like, the index may be directly formed on the carrier by laser cutting or a diamond cutter. However, the former is preferable from the viewpoint of detection. Preferably, the index is to form an opening of a partition wall formed on the carrier.

Further, in the case of a chip in which a plurality of probes are two-dimensionally arranged and fixed, it is preferable that the fixing regions partitioned by such partition walls are arranged in a matrix as shown in FIG. . In this case, the index of the present invention is
By providing in a non-fixed region with a shape different from the member forming the partition wall, the position is accurately adjusted by the index, and by the partition wall, the position of the discharge target due to the unexpected position change of the discharge liquid due to the flow of the air current, etc. It is possible to suppress the overflow and reliably apply the solution to the opening. Then, since only the portion existing as an index needs to be detected by a detection means or the like, a method of performing alignment after detecting positional information of the entire chip (for example, visually or by detecting the shape of the partition wall with a CCD or the like), etc. Compared with, the alignment adjustment can be performed in a shorter time, and the remarkable effect of further shortening the chip manufacturing time can be obtained.

The partition wall is preferably made of a resin composition or a metal composition, and the resin composition or the metal composition is preferably light-shielding. A method for manufacturing an index having such a partition will be described later in detail.

At least one index is provided for each probe array. A DNA chip is formed by assembling some of these probe arrays. Further, from the viewpoints of alignment between the head and the carrier when ejecting the probe solution by the inkjet method, and accurate and rapid position detection of the target substance, it is preferable to provide a plurality of the above-mentioned indicators on each chip. . By scanning two or more indexes, two-dimensional information on the chip can be obtained more accurately, and when the ejection head has an inaccurate inclination with respect to the carrier, the ejection head and the inclination can be adjusted. It will be possible. Further, when a plurality of indexes are provided, it is not necessary that they all have the same shape, and a plurality of indexes having different shapes may be used.

As shown in FIG. 1, the position for forming the index of each probe array can be provided at any position other than the probe fixing region (which means a specific place on the carrier for fixing the probe). As shown in FIG. 1, it may be provided in the peripheral portion of the chip, or in the case where there is a partition wall between the adjacent probe-probe regions or the carrier, it may be provided in the partition wall between the openings (wells). Is also good. When it is provided in the adjacent probe-probe region, the probe fixing region can be expanded to the peripheral portion of the chip,
Since more probes can be mounted on each chip, high density can be achieved. Further, when forming the index on the peripheral portion of each chip, it is preferable to form the index on the peripheral portion as close as possible to the probe fixing region from the viewpoint of accurate positioning.

The shape of the index can be any shape, but it serves the purpose of aligning the head and the carrier and detecting the position of the target substance when the probe is ejected accurately and quickly by using the index. Is good. Any of a cross shape, a circle, a square, a rectangle, an isosceles triangle, a butterfly shape and the like can be used. The cross shape is preferable because the position of the center point can be easily identified and the shape is easy in manufacturing. Horizontal line width 30μm, length 150μm and vertical line width 30μ
A cross-shaped index having a length of m and a length of 150 μm is preferable.

The size of the index can be appropriately changed depending on the accuracy of positioning and position detection. When the partition wall is provided on the carrier, the size of the index can be typically set to the same size as the area occupied by each opening (well). The thickness of the index is the same as that of the partition when it is formed at the same time as the partition, and is preferably 0.5 μm.
It can be selected from the range of up to 100 μm.

The above-mentioned alignment used in the method for producing a probe carrier of the present invention uses at least one index for both the alignment of the head and the carrier during the preparation of the probe carrier and the positioning during the detection of the target substance. Can be used for
It is not necessary to form an index for each of both uses, and the probe non-fixed region can be reduced as long as this is achieved. The index can also be used as a mark for confirming the front and back of the carrier.

Further, in this case, regarding the immobilization of the probes, it is not always necessary to attach the probes to all the wells, and the number, types and positions of the probes can be appropriately changed depending on the use purpose. It is also preferable that the information about the fixed location at this time can be confirmed by this index. For example, depending on the number and type of probes to be immobilized, change the shape and number of indicators,
It is also a preferable embodiment to adopt a configuration in which probe position information at the time of detection is obtained.

Further, when the partition wall is provided on the carrier, if the member forming the partition wall has a light shielding property and the index is the opening of the partition wall, the partition wall portion is shielded from light during detection by fluorescence detection or the like. Even in the case of measurement in a dark room or the like, the function as an index can be sufficiently maintained. Furthermore, by providing a material suitable for the measurement conditions on the opening, such as coating the opening of the light-shielding member with a fluorescent substance, it is possible to perform alignment adjustment regardless of the adjustment environment.

Hereinafter, with reference to the drawings, a probe-fixing carrier of the present invention, a probe-supporting probe carrier of the present invention, and a manufacturing method thereof, particularly when the probe non-fixing region has a partition, will be described with reference to embodiments. To do.

FIG. 2 is a process chart schematically showing a method for producing a probe carrier according to the present invention. Each step will be described below. In FIG. 2, 11 is an index, 12 is a partition having an index, 13 is a well, 21 is a carrier, 22 is a partition wall layer preferably made of a resin composition or a metal composition, 23 is a photoresist mask, and 24 is a probe solution discharge. Head and 25 are probe solutions. Figure 1 (a)
This is an example of the present embodiment, and the index 11 has a cross shape,
Surrounded by the partition wall 12, a concave opening is formed. In addition, in FIG. 1A, the center of the cross and the center of the opening are illustrated so as to overlap for convenience, but the present invention is not limited to these.

Step (a): Carrier for probe array Carrier 21 for fixing probes is prepared (see FIG. 2B). The "carrier" for the probe array is one that carries the above-mentioned probe, and can hold the probe,
Moreover, it is not particularly limited as long as it does not interfere with the detection of the target substance. The carrier includes, in addition to a glass substrate, a silicon substrate, a metal substrate, an acryl, a resin substrate, a hollow body and a tubular carrier, and each carrier may be subjected to an appropriate surface treatment such as hydrophilizing the surface. I do not care. As a substrate to be used for optically detecting the reaction, an optically transparent substrate or an optically black substrate is desirable in some cases. Such desirable carriers include synthetic quartz, glass substrates such as fused quartz, silicon wafers,
In addition, resin substrates such as acrylic, polycarbonate, polystyrene, vinyl chloride, etc.
A substrate mixed with a dye can be used. As the black pigment, carbon black or an organic black pigment can be used. On the surface of the carrier, a structure that reacts with various organic functional groups introduced into the probe to form a covalent bond,
It is desirable to perform a treatment for introducing an organic functional group in advance. As a preferred combination of the functional group introduced at the probe end and the functional group introduced on the carrier surface capable of binding to the functional group, a combination of thiol (-SH) (nucleic acid probe end) and a maleimide group (carrier surface) , A combination of an amino group (terminal of nucleic acid probe) and an epoxy group (carrier surface), and the like.

The surface of the carrier 21 may be subjected to surface treatment such as plasma treatment, UV treatment, coupling treatment, albumin treatment and the like.

Step (b): Preparation of resin composition layer Next, a partition wall layer 22 for forming the partition wall 12 for forming the well 13 and the index 11 which are openings on the carrier 21.
Are formed (see FIG. 2B). The partition 12 is
When it is a black light-shielding layer, it is called a black matrix.

The partition wall 12 is preferably a light-shielding layer that shields light between adjacent probe fixing regions, and in this case, it may be a black matrix as described above or a black stripe. The use of the light-shielding layer can further improve the detection accuracy (SN ratio) in the case of optically detecting the hybridization between the probe and the target substance on the carrier (for example, detecting fluorescence). it can.

As the constituent material of the partition layer 22, for example,
It may be a metal such as chrome, aluminum or gold. In particular, black chrome has a high light-shielding property, which is desirable from the viewpoint of reliability when optical detection is performed in combination with a transparent substrate. However, in the case of metal,
If anything, it is highly hydrophilic, and considering the uniformity of the film thickness, etc., a film thickness of several thousand angstroms is common when forming a film by means such as vapor deposition, so consider this point. It is good to use.

Further, as a preferable constituent material of the partition wall layer 22, a material having a relatively high hydrophobicity as compared with the substrate is preferable, and, for example, an epoxy resin, an acrylic resin, a polyimide-based material including polyamide-imide is used. A photosensitive or non-photosensitive resin material such as resin, urethane resin, polyester resin, or polyvinyl resin can be used. Two
It is preferable to have a heat resistance of 50 ° C. or higher, and from this point, epoxy resin, acrylic resin, and polyimide resin are preferably used.

When the partition wall 12 is used as a suitable light-shielding layer, the resin composition layer 22 is formed by using a black resin composition in which a light-shielding agent is dispersed in the resin composition. As the light-shielding agent, as will be described later, it is desirable to use carbon black in order to obtain high water repellency of the partition wall 12 and appropriate surface roughness. Examples of the carbon black include channel black, roller black and disc black. Manufactured by the so-called contact method,
Gas farnest black, those produced by the farnest method called oil farnest black, thermal black, those produced by the thermal method called acetylene black can be used, but in particular, Channel black, gas farnest black and oil farnest black are preferred. It may contain a black organic pigment. Further, a black resist which is generally commercially available can also be used.

The partition layer 22 can be formed by a method such as spin coating, roll coating, bar coating, spray coating, dip coating, or a printing method.

When the partition wall 12 is not formed on the carrier, this forming step is omitted.

Step (c): Partition and alignment production index 11 and the partition 12 forming the well 13 are simultaneously formed by photolithography (see FIG. 2C).

As one method for forming the pattern of the partition wall 12 and the index 11, a photoresist mask 23 having the pattern of the well 13 and the index 11 is formed on the resin coated on the surface of the carrier 21 in the step (b). There is a method of forming a partition wall 12 having a plurality of wells 13 which are probe fixing regions and a concave index shape by selectively exposing and developing and patterning. The details of the shape, arrangement, number, etc. of the index 11 have been described above, but the index can be produced by using a photoresist mask corresponding to such index. Further, if it is a photosensitive resin, it is possible to perform patterning by a photolithography process using the resin itself as a photomask. As such photosensitive resin, UV resist, DEEP-U
A V resist, an ultraviolet curable resin or the like can be used.
Examples of the UV resist include a cyclized polyisoprene-aromatic bisazide-based resist, a negative resist such as a phenol resin-aromatic azide compound-based resist, and a positive resist such as a novolak resin-diazonaphthoquinone-based resist. In addition, it is preferable that the partition walls of the black matrix are made water-repellent by performing dry etching or the like described later after patterning. When a photosensitive material is used as the resin composition layer 22, the pattern formed by patterning the resin layer 22 can be further fired to improve the water repellency of the partition wall 12. By imparting water repellency, the probe solution can be smoothly applied to a desired well even if some displacement occurs in applying the probe solution to the opening. The opening 13 is usually formed as a well.

Partition opening 1 corresponding to the probe fixing region
The shape of 3 can be selected in consideration of easiness in forming, handleability, operability in detection when performing detection, and is not limited to various polygonal shapes, elliptical shapes, etc.
A simple shape such as a square, a rectangle, or a circle is desirable, and the arrangement of the openings on the carrier can be appropriately changed as desired.

The area occupied by the probe solution applied to one opening (well) 13 is 0.01 (for example, 0.1).
μm × 0.1 μm) μm 2-40,000 (for example, 200
(μm × 200 μm) μm2 is generally used,
The area is determined by the size of the array itself and the density of the array matrix. Therefore, the size and volume of one opening (well) can be set by the area occupied by the probe solution. The depth of the opening (well) depends on the method of manufacturing the opening (well), but is preferably selected in the range of 0.5 μm to 100 μm. The volume is determined by the area and depth of these openings (wells). As a method for manufacturing such an opening (well), for example, the method described in JP-A-11-099000 can be used as it is.

The partition wall 12 may have a rectangular partition wall cross section as shown in FIG. 4 (partition wall 42), or may have a trapezoidal partition wall cross section as shown in FIG. 5 (a) (partition wall. 52).
FIG. 5B is an enlarged view of a part of the partition wall cross section of FIG. Further, as shown in FIG. 6, the partition wall cross section may have an inverted trapezoidal shape (partition wall 62).

In the sectional view of the partition wall of FIG. 5B, A1 and A2 are 8 which is the height from the bottom of the partition wall section to the top of the partition wall.
The partition walls at heights of 0 to 100%, B1 and B2 indicate the portions where the partition wall cross section and the carrier come into contact. L1 is A1 and A
2 is the interval, L2 is the interval between B1 and B2, T is the height of the top of the partition, and T ′ is 8 the height of the top of the partition.
The height is 0 to 100%. Further, the sections A1 and B1 and A2 and B2 are defined as the side surfaces of the partition wall in the present invention.

Here, the length L1 in the direction parallel to the substrate at the partition wall upper portion at a height T'between 80% and 100% of the height T of the partition wall uppermost portion from the bottom wall of the partition wall 52 is defined. When the average value is a and the length L2 of the lower bottom in the cross section of the partition wall 52 is b, the relationship between a and b is expressed by the formula 1.3 ≧ a.
The shape represented by /b≧0.7 is preferable. a / b is 0.
When it is less than 7, unevenness occurs in the probe of the probe solution,
It is not preferable from the viewpoint of the uniformity of the probe, and the problem of white spots occurs. On the other hand, as shown in FIG.
b can be larger than 1, and the partition wall 62 has an inverted trapezoidal cross section. In this case, the probe solution enters the bulky portion of the partition wall 62, which is more preferable because the area of the "white spot" portion is narrowed at the time of detection. However, when the formula a / b becomes large, the structure becomes unstable, which is not preferable. Formula a / b is preferably 1.3 or less.

Further, the height of the partition wall 3 from the surface of the carrier 1 is preferably 0.5 μm to 100 in consideration of the method of forming the partition wall, the capacity of the opening, the amount of the reaction system solution to be supplied, and the like.
It can be selected from the range of μm. Particularly when the partition walls 3 are formed by photolithography, it is desirable that the thickness be 20 μm or less.

When the partition wall 12 is not formed on the carrier, this forming step may be omitted and a desired index may be produced. As described above, a concave projection or the like may be provided on the carrier by using a photolithography method using a resin material or the like. When the carrier 21 is a glass substrate or the like, laser cutting or diamond cutter, etc. Thus, a concave index can be formed directly on the carrier.

Step (d): Dry etching treatment It is desirable to perform dry etching treatment on the carrier 21 on which the partition wall 12 is formed. (See FIG. 2D) That is, low-pressure plasma in which a gas containing at least one selected from oxygen, argon, and helium is introduced, and the carrier 1 is irradiated with plasma under a reduced-pressure atmosphere or an atmospheric pressure atmosphere. Processing or atmospheric pressure plasma processing.

By performing the dry etching treatment, contaminants attached to the surface of the carrier 21 in the step of forming the partition wall 12 are removed, and the surface is cleaned to improve the affinity for the probe solution 25 in the subsequent step. The water treatment process performed in the process allows the probe solution 25 to be better diffused in the opening 13. Further, the dry etching treatment roughens the surface of the partition wall 12 and improves the water repellency.

Step (e): Plasma treatment Carrier 21 which is dry-etched if necessary
Then, plasma treatment is performed by plasma irradiation in a gas atmosphere containing at least fluorine atoms. (See FIG. 2E) By the plasma treatment, fluorine or a fluorine compound in the introduced gas enters the surface layer of the partition wall 12 and the partition wall 12
The water repellency of the surface layer increases.

Particularly when the partition wall 12 is made of a resin composition containing carbon black, extremely high water repellency is exhibited. It is considered that the reason is that the carbon black is exposed on the surface of the partition wall 12 by the dry etching treatment in the previous step (d) and fluorine or a fluorine compound is bonded to the carbon black by the plasma treatment in this step. Therefore, in the present invention, the partition wall 12
It is desirable to include carbon black in the.

As the gas containing at least fluorine atoms, which is introduced in this step, CF ₄ , CHF ₃ and C are used.
It is preferable to use at least one halogen gas selected from ₂ F ₆ , SF ₆ , C3F ₈ , and C ₅ F ₈ . In particular, C
₅ F ₈ (octafluorocyclopentene) has an ozone depletion potential of 0 and at the same time has an atmospheric life of 0.98 as compared with conventional gases (CF ₄ : 50,000 years, C ₄ F ₈ : 3200 years).
Very short in years. Therefore, the global warming potential is 90 (CO
And ₂ = 2 was 100 years accumulated value), as compared with the conventional gas _{(CF 4: 6500, C 4} F 8: 8700) very small, it is very effective in ozone layer and global environmental protection, in the present invention Desirable for use.

Further, as the introduced gas, if necessary,
You may use together gas, such as oxygen, argon, and helium.
In the present invention, the CF_Four, CHF_Three, C2F₆,
SF ₆, C_ThreeF₈, C₅F₈Halogen gas selected from
One or more types and O_TwoIf you use a mixed gas of
Therefore, the degree of water repellency can be controlled. However,
O in the mixed gas_TwoThe mixing ratio of exceeds 30%
And O_TwoOxidation reaction due to becomes dominant and water repellency improving effect
Because the fruit is blocked, O_TwoMixing ratio exceeds 30%
If this happens, the damage to the resin becomes significant, so
O when using mixed gas_TwoThe mixing ratio of 30% or less
Must be used in range.

As a method of generating plasma in this step and the previous dry etching treatment step, a method such as low frequency discharge, high frequency discharge, microwave discharge or the like can be used, and pressure, gas flow rate, discharge during plasma irradiation frequency,
Conditions such as processing time can be set arbitrarily.

FIG. 7 and FIG. 8 show schematic views of a plasma generator which can be used in the dry etching process and the plasma process of the present invention. In the figure, 71 is an upper electrode, 72 is a lower electrode, 73 is a carrier to be treated, and 74 is a high frequency power source. The apparatus applies a high frequency voltage to the parallel plate bipolar electrodes to generate plasma. FIG. 7 shows a cathode coupling type device, and FIG. 8 shows an anode coupling type device. In both types, the partition wall 12 is selected depending on conditions such as pressure, gas flow rate, discharge frequency, and treatment time.
The water repellency of the surface, the surface roughness, and the parent probe solution property of the surface of the carrier 21 can be set to desired levels.

In the plasma generator shown in FIGS. 7 and 8, the cathode coupling method of FIG. 7 can shorten the dry etching processing time, which is advantageous for the processing step. Further, the anode coupling method of FIG. 8 is advantageous in that the carrier 21 is not damaged more than necessary. Therefore, the plasma generator used in the dry etching treatment step and the plasma treatment step is
It may be selected according to the material of the carrier 21 and the partition wall 12.

By this step, the amount of the fluorine compound existing on the surface of the carrier 21 exposed in the opening 13 of the partition 12 is halved.
The surface roughness of the surface is also reduced as compared with that before the step.

In this step, when the carrier 21 is brought into contact with water and then heated and dried at a high temperature of more than 100 ° C., the spreadability of the probe solution may decrease, so that the drying step is 100 times. It is desirable to carry out at a temperature of ℃ or less.

Step (f): Water Treatment A water treatment step of bringing water into contact with the surface of the carrier 21 subjected to the plasma treatment is carried out to improve the spreadability of the probe solution in the opening 13 of the partition 12. (See FIG. 2F) As a result, the probe solution can be sufficiently spread in the opening 13 even when a small amount of the probe solution is applied.

The water used in this step is preferably pure water. Further, as a method for bringing the support carrier 21 into contact with water, water and the carrier 2 may be immersed in water, washed with water, or the like.
There is no particular limitation as long as it is a method of bringing 1 into contact completely. However, in the case where the pattern on the support carrier 21 has a complicated pattern shape, in particular, since the support carrier 21 is sufficiently contacted with water at a fine portion such as a boundary portion between the partition wall 12 and the opening 13 and a corner portion, It is preferable to immerse in water and simultaneously irradiate with ultrasonic waves, or wash with fine, high-pressure water droplets.

Further, the temperature of the water to be brought into contact with the carrier 21 is preferably high from the viewpoint of effectively modifying the surface of the opening 13, but in consideration of the economic effect of heating the water, the temperature is 20 to 60. The range of ° C is preferred.

The water repellency and hydrophilicity of the partition wall and the probe fixing region (opening) after the above process treatment will be described below. In the present invention, the partition walls are formed of a resin composition containing carbon black, and the water repellency of the partition wall surface can be increased to 120 ° or more after the plasma treatment and the water treatment step. Of 12 surfaces,
The degree of water repellency after the water treatment step is such that the contact angle measured with pure water is 120 ° or more, preferably 135 ° or more, more preferably 150 ° or more, and most preferably 180 °. When the contact angle is less than 120 °, liquid mixture is likely to occur,
A large amount of probe solution cannot be applied. In particular, when manufacturing a probe carrier, it becomes difficult to manufacture a probe carrier having high color purity. In the conventional method, it is difficult to make the water repellency of the partition wall surface at a contact angle of 120 ° or more, and PTF used as a highly water repellent material.
110 ° even for E (polytetrafluoroethylene)
It was weak. By setting the water repellency of the partition surface to a contact angle of 120 ° or more, it is possible to effectively prevent the mixture of probe solutions.

Regarding the hydrophilicity of the opening 13 of the carrier 21, the contact angle measured with pure water is preferably 30 ° or less. 30 ° contact angle for probe solution
By the following, the carrier 2 exposed in the opening 13 by the dry etching treatment in the previous step (d)
(1) The contaminants attached to the surface can be removed, and the effect of improving the ink spreadability by the water treatment performed in the subsequent step can be sufficiently obtained. In particular, it is desirable that the angle be 20 ° or less.

Regarding the water repellency of the partition wall and the affinity of the probe fixing region for the probe solution in the opening 13 of the probe fixing carrier 21 of the present invention produced as described above, the contact angle for each probe solution is the same. It depends on the solvent contained in the solution. A solution preferably used as a solvent for the probe solution is glycerin 7.5w.
t%, urea 7.5 wt%, thiodiglycol 7.5 wt
%, General formula (IV):

[0100]

[Chemical 1]

1 wt% of acetylene alcohol represented by
An aqueous solution containing is preferably used. (In the above formula (I),
R1, R2, R3 and R4 represent 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 and n.
= 0 or 1 ≦ m + n ≦ 30 and m + n = 1
In the case of, m or n is 0. ). Regarding the water repellency of the partition wall 12 with respect to the probe solution when the above probe solution is used, the present invention produced by forming the partition wall 12 from a resin composition containing carbon black, performing dry etching treatment, and then performing plasma treatment. In the probe fixing array according to the above, the contact angle with respect to the probe solution is 9
The angle can be set to 0 ° or more, and in this range, the probe solution has sufficient water repellency, and it is possible to effectively prevent liquid mixture and white spots. It is more preferably 100 ° or more, more preferably 115 ° or more, and 13
It is more preferably 0 ° or more. Further, regarding the affinity of the partition wall 12 for the probe solution, the contact angle with respect to the probe solution is preferably 30 ° or less. In this range, the effect of improving the ink spreading property by water treatment can be sufficiently obtained. In particular, it is desirable that the angle be 20 ° or less.

Step (g): Application of probe solution to carrier and immobilization Probe solution is applied to and immobilization on the carrier. After adjusting the position of the head with respect to the carrier (drawing alignment), an ink jet type ejecting apparatus is used. Using, the probe solution 25 is applied from the inkjet head 24 into the well 13 of the partition 12 (see FIG. 2G). Hereinafter, one suitable embodiment regarding application and immobilization of the probe solution on the carrier will be described in detail.

(Drawing Alignment) In the drawing alignment, the index produced as described above is imaged by an image pickup means such as a CCD camera using a laser beam as a light source, processed by an image processing device, recognized, and its position coordinate is determined. Based on the detection result, the XYθ stage provided in the liquid ejection apparatus is adjusted to align the head and the carrier. When the size of the index is reduced or the line width of the index is narrowed, the area sensor of the CCD camera used for positioning has a large number of pixels, or a CCD camera having a plurality of area sensors arranged therein. Alignment may be performed using.

(Liquid Ejecting Device) The probe carrier manufacturing device used in the method of the present invention comprises a liquid ejecting means used for ejecting a probe solution by an ink jet method, and for fixing a probe having an index in the probe non-fixing region. A probe carrier manufacturing apparatus capable of applying a solution containing a probe to a specific position on a carrier with reference to the position of the index.

More specifically, the probe carrier manufacturing apparatus used in the method of the present invention comprises a liquid ejecting means used for ejecting the probe solution by an ink jet method. As the liquid ejecting mechanism, the probe solution is used. A liquid storage portion for storing the liquid storage portion, a discharge port provided corresponding to each of the liquid storage portions, a liquid path for communicating the liquid storage portion and the discharge port in common, and a liquid passage corresponding to each of the discharge ports. A liquid ejecting section provided with ejecting energy generating means capable of ejecting an independent probe solution by each of the ejecting ports, and at least a number of liquid ejecting sections corresponding to the plurality of kinds of probe solutions are provided. , A structure capable of detecting the position of the index, and corresponding to the position where the probe is arranged after aligning the head and the carrier to a desired position with the index as a reference It is possible to eject each of the solutions of the plurality of types of probes from the liquid ejecting device based on the information regarding the arrangement position while applying relative movement to the carrier having the concave portion on the surface, and apply the solution onto the carrier. preferable. As a configuration capable of detecting the position of the index, it is preferable to have an image pickup means such as a CCD camera for picking up an image of the probe array carrier surface, an image processing means, and an XYθ stage for position adjustment by drawing alignment.

FIG. 9 is a block diagram of the controller of the probe carrier manufacturing apparatus 101. Reference numeral 95 is an interface for exchanging data with the teaching pendant 92, 96 is a CPU for controlling the probe carrier manufacturing apparatus 101 and image processing, 97 is a ROM storing a control program for operating the CPU 96, and 98 is a ROM. A RAM storing abnormal information and the like, 99 an ejection control unit for controlling the ejection of the probe solution, 100 a stage control unit for controlling the operation of the XY stage (not shown) of the color filter manufacturing apparatus 101, and 70 connected to the controller 91. And a probe carrier manufacturing apparatus that operates according to the instruction.

(Probe Solution Ejection Head) The liquid ejection head used in the method of the present invention is a head for ejecting the probe solution by utilizing thermal energy, and is for generating thermal energy to be applied to the probe solution. It is preferable to provide the heat energy generator of. Inkjet methods that can be used in the manufacture of probe carriers include thermal jets that eject liquid by generating bubbles using thermal energy generated from electrothermal converters such as heaters and laser light. There are a system (a bubble jet (registered trademark) system) and a piezo inkjet system in which a liquid is ejected by the deformation of the device caused by applying a voltage to the piezo device, and any of them can be used in the method of the present invention. Of these, the head for the thermal jet method has a simpler structure than the head for the piezo inkjet method,
It is suitable for downsizing the head and increasing the number of nozzles. As mentioned above, in the case of the thermal jet method, the binding reaction to the carrier is completed in a short time, and the secondary structure of DNA is also eliminated by heat. The thermal jet method is an inkjet method more preferably used in the present invention in that the efficiency of the subsequent hybridization reaction can be increased.

With regard to its typical structure and principle, see, for example, US Pat. Nos. 4,723,129 and 4740.
It is preferable to use the basic principle in the field of ink recording disclosed in Japanese Patent No. 796. This method can be applied to both the so-called on-demand type and the continuous type. In particular, in the case of the on-demand type, it is arranged corresponding to the sheet or liquid path holding the liquid (ink). The electrothermal converter being subjected to a rapid temperature rise corresponding to the recorded information and exceeding the nucleate boiling.
By applying two drive signals, heat energy is generated in the electrothermal converter, causing film boiling on the heat acting surface of the recording head (liquid ejection head), and as a result, one-to-one correspondence with this drive signal. This is effective because bubbles can be formed in the formed liquid (ink). The liquid (ink) is ejected through the ejection openings by the growth and contraction of the bubbles to form at least one droplet. If this drive signal is made into a pulse shape, the growth and contraction of bubbles will be performed immediately and appropriately,
In particular, it is possible to achieve ejection of liquid (ink) with excellent responsiveness,
More preferable. As the pulse-shaped drive signal, U.S. Pat. Nos. 4,463,359 and 4,345,262 are used.
Those described in the specification are suitable. If the conditions described in US Pat. No. 4,313,124 of the invention relating to the rate of temperature rise on the heat acting surface are adopted, more excellent recording can be performed.

As the constitution of the recording head, in addition to the combination constitution of the discharge port, the liquid passage, and the electrothermal converter (the linear liquid passage or the right-angled liquid passage) as disclosed in the above-mentioned respective specifications, U.S. Pat. No. 4,558,333 and U.S. Pat. No. 44, which disclose a configuration in which a heat acting portion is arranged in a bending region.
The structure using the specification of No. 59600 is also included in the present invention. In addition, Japanese Unexamined Patent Publication No. 59-123670 discloses a configuration in which a common slit is used as a discharge portion of the electrothermal converter for a plurality of electrothermal converters, and an opening for absorbing a pressure wave of thermal energy is provided. The effect of the present invention is effective even when the configuration corresponding to the ejection portion is disclosed in JP-A-59-138461. That is, regardless of the form of the recording head (liquid ejection head), according to the present invention, spotting of the probe can be performed reliably and efficiently.

Furthermore, the present invention can be effectively applied to a full-line type liquid discharge head having a length corresponding to the maximum width of the carrier that can be spotted by the liquid discharge device. Such a liquid ejection head may have a configuration that fills its length by a combination of a plurality of heads or a configuration as one head that is integrally formed.

In addition, even in the case of the serial type, when it is attached to the head fixed to the apparatus main body or the apparatus main body, it becomes possible to electrically connect to the apparatus main body and apply the probe solution from the apparatus main body. The present invention is also effective when a replaceable chip type liquid discharge head or a cartridge type head in which a solution reservoir is integrally provided in the liquid discharge head itself is used.

Further, as the structure of the liquid ejecting apparatus, it is preferable to add an ejection recovering means for the head, a preliminary auxiliary means, etc. because the effects of the present invention can be further stabilized. Specific examples thereof include cleaning means for the liquid ejection head, pressurizing or suctioning means, preheating means for heating using an electrothermal converter or a heating element other than this, or a combination thereof, Preliminary ejection means for performing ejection other than spotting can be mentioned.

The most effective of the above solutions is
The film boiling method described above is executed.

(Probe Solution) The composition of the probe solution and the ejection method used when the probe carrier of the present invention is manufactured will be described in detail below.

In the present invention, the probe immobilized on the carrier is capable of specifically binding to a specific target substance. Furthermore, this probe includes an oligonucleotide or polynucleotide that can be recognized by a specific target,
Alternatively, other polymers are included. The term “probe” means a probe molecule itself having a probe function such as an individual polynucleotide molecule,
It may mean a population of probe molecules having the same probe function, such as polynucleotides of the same sequence, which are surface-immobilized at dispersed positions, and a molecule often referred to as a ligand is also included. Also, probe and target are often used interchangeably, and the probe is one that can or will become capable of binding to the target as part of a ligand-antiligand (sometimes called a receptor) pair. is there. The probes and targets in the present invention may include bases as found in nature, or analogs thereof.

[0116] As an example of the probe supported on the carrier, a part of an oligonucleotide consisting of a base sequence capable of hybridizing with a target nucleic acid has a binding part with the carrier via a linker. One having a structure in which it is connected to the surface of the carrier at the bonding portion of. The positions of the carrier and the binding site in the molecule of the oligonucleotide in the case of such a structure are not limited as long as the desired hybridization reaction is not impaired.

The probe employed in the probe carrier produced by the method of the present invention is appropriately selected according to the purpose of use, but in order to suitably carry out the method of the present invention, As the probe, DNA, RN
A, cDNA (complementary DNA), PNA,
Oligonucleotides, polynucleotides, other nucleic acids, oligopeptides, polypeptides, proteins, enzymes, substrates for enzymes, antibodies, epitopes for antibodies, antigens, hormones, hormone receptors, ligands,
It is preferably any one of a ligand receptor, an oligosaccharide and a polysaccharide, and if necessary, two or more kinds of these can be used in combination.

In the present invention, a probe carrier is prepared by immobilizing plural kinds of these probes on independent regions, for example, dot-shaped spots on the carrier surface (including the inner wall surface of the hollow body or tubular carrier). What is arranged at a predetermined interval is called a probe array.

On the other hand, the probe has a structure capable of binding to the surface of the carrier, and it is desirable that the probe is immobilized on the carrier via this structure capable of binding. At that time, the structure capable of binding to the carrier surface of the probe is an amino group, a mercapto group, a carboxyl group, a hydroxyl group, an acid halide (haloformyl group; -COX), a halide (-X), an aziridine, a maleimide group, a succinimide. Group, isothiocyanate, sulfonyl chloride (-S
O ₂ Cl) group, aldehyde (formyl group; —CHO)
It is preferably formed by a treatment of introducing an organic functional group such as a group, hydrazine, or iodoacetamide. Further, depending on the structure required for binding to the probe-side carrier, the surface of the carrier may be subjected to the necessary treatment as described above.

When a probe solution containing a probe is applied onto a carrier as described above by an inkjet method, the liquid composition should be adjusted so that the liquid droplets do not unnecessarily spread on the carrier and remain at a predetermined position. Is preferred. The liquid composition preferably does not impair the original performance of the probe and does not impair the reactivity with the functional group on the surface of the carrier introduced into the probe.

When the inkjet method is used, since the droplets applied to the surface of the carrier are minute, in order to prevent evaporation and drying after application, the carrier is placed in a reaction container such as a moisturizing container during the reaction. It is a preferable method to store the above, but it is also effective to include a moisturizer in the liquid to be ejected. In particular, in the thermal jet method, the presence of the moisturizing component and the surface tension adjusting component is important because the temperature is increased during the ejection. As such a marker substance, or as a solvent that imparts the probe to the carrier surface,
5-10 wt% urea, 5-10 wt% glycerin,
Those containing 5 to 10 wt% of thiodiglycol and 1 wt% of acetylene alcohol can be preferably used.

Considering the ink jet discharge characteristics of the liquid, the stability of the probe in the liquid and the thermal jet (Bubble Jet (registered trademark)) discharge, the probe solution has, for example, 2 mer to 5000 me in the liquid.
r, especially a 2mer to 60mer nucleic acid probe,
It is preferably contained at a concentration of 0.05 to 500 μM, particularly 2 to 50 μM.

As a liquid characteristic of the probe solution, a thermal jet head (bubble jet (registered trademark) head) is used.
From the viewpoint of dischargeability from, for example, its viscosity is 1
-15 cps and a surface tension of 30 dyn / cm or more are preferable. The viscosity is 1 to 5 cps and the surface tension is 30 to 50.
When it is set to dyn / cm, the landing position on the carrier becomes extremely accurate, and it is particularly preferably used.

It should be noted that a structure such as a well is formed on the surface of the carrier so as to prevent the liquid applied on the carrier by the liquid discharge head from spreading on the carrier and mixing with adjacent spots. In the case where the above is provided, the viscosity and surface tension of the liquid, and the base length and concentration of the nucleic acid probe can be used even if they are out of the above ranges.

(Discharge Mode) The amount of the probe solution discharged from one nozzle at a time is appropriately selected according to the areal density of each probe spot on the probe carrier. It is selected according to the dot size and shape of the array to be formed, taking into consideration various factors such as the affinity of the carrier and the reactivity between the probe and the carrier. In this case, the amount of discharged liquid per time is 0.1 to
100 pl (picoliter), preferably 0.1-50
It can be selected from the range of pl. In order to increase the density of the probe, it is preferable that the amount discharged at one time is as small as possible, but if the amount discharged is extremely small, it will not reach the carrier due to air resistance. The discharge amount per discharge is preferably 0.1 to 10 pl, more preferably 0.1.
It is preferably up to 5 pl, and it is preferable to design and adjust the nozzle diameter and the like according to the liquid amount.

In order to set the density of the nucleic acid probe on the carrier to the above value (for example, 10000 or more spots per inch, the upper limit is about 1 × 10 6 spots), the spot diameter of each nucleic acid probe is For example, 20 to 100 μm
It is preferable that the spots are in the order of magnitude and that the spots are independent of each other. Then, such a spot is determined by the characteristics of the liquid ejected from the bubble jet (registered trademark) head, the surface characteristics of the carrier to which the liquid adheres, and the like.

In the present invention, a solution of a target substance capable of specifically binding to a probe is supplied to these probe carriers and placed under appropriate reaction conditions for binding. When it is necessary to supply solutions of different target substances to individual spots, at least one solution of at least one target substance to be bound to the probe is supplied to each of the plurality of spots of the probe carrier. . Further, when supplying these target substances to the probe carrier by ejecting the liquid as described above, it is possible to quantitatively supply a small amount of liquid and to adjust the alignment between the substrate and the ejection head. Also, the above-mentioned index can be used as a reference and is preferable.

A method for detecting the position of the target substance specifically bound to the probe immobilized on the probe carrier of the present invention produced as described above will be described below with reference to the above-mentioned index. .

The indicator and the target substance are preferably labeled with a radioactive label or a fluorescent label, and for detecting the position of the target substance, particularly when the carrier has a partition wall and an opening (well), the target and the target substance are specifically labeled with the probe. It is preferably performed by detecting the fluorescence emitted by the fluorescent label associated with the bound target substance.

In the alignment of the scanned image at the time of detection,
The position of the fluorescence generated by the fluorescent marker is detected by imaging the image with an image pickup device such as a CCD camera using the laser light as a light source, processing the image with an image processing device, recognizing the position coordinate of the index, and using the position coordinate as a reference. By detecting, it is possible to identify the probe that fluoresces among the probes formed in advance with reference to the index position coordinates. If the index is small or the line width of the index is narrow, use a sensor with a large number of pixels in the area sensor of the CCD camera used for positioning, or use a CCD camera with multiple area sensors arranged for alignment. Should be done.

It is preferable to employ a method in which information on unnecessary substances remaining on the black matrix is not detected at the time of detection, because the step of surface treatment such as albumin treatment can be simplified. For example, a method of irradiating light from a light source such as a laser beam on the back surface of the carrier to detect fluorescence from the fluorescent material, or a method of detecting fluorescence from the fluorescent material from the back surface can be mentioned. More specifically, the carrier is a carrier made of a light-transmissive plate, and has a partition wall for partitioning a specific position where a plurality of types of probes are fixed on one surface of the carrier. Then, the fixed position of the probe can be suitably specified by detecting the fluorescence penetrating the carrier emitted by the radioactive label under the predetermined condition on the other surface of the carrier.

[0132]

Example (Example 1) Glass substrate (made by Corning "1"
737 ") is coated with a black resist containing carbon black (" CK-A143B resist "manufactured by FUJIFILM Ohlin), predetermined exposure by a UV exposure device, development with an inorganic alkaline aqueous solution, and 2 in a clean oven.
After heating at 20 ° C for 60 minutes to perform post-baking,
A black matrix pattern (partition wall) having a rectangular opening with a film thickness of 2 μm and 100 μm × 200 μm was produced. Along with the black matrix pattern, the horizontal line width is 30 μm, the length is 150 μm, the vertical line width is 30 μm, and the length is 1.
A cross-shaped index consisting of 50 μm is placed in two corners around the chip.
Individually formed (see FIG. 1).

Next, the solvent for ejecting the fluorescent dye Rhodamine B with the liquid ejection head, that is, glycerin 7.5 wt%, urea 7.5 wt%, thiodiglycol 7.5 wt%, general formula (IV):

[0134]

[Chemical 2]

An aqueous solution containing 1 wt% of acetylene alcohol represented by (for example, trade name: acetylenol EH Kawaken Fine Chemical Co., Ltd.) was dissolved at a concentration of 10 μM. This dye solution was injected into every other liquid reservoir formed by being connected to each nozzle of the inkjet head. A fluorescent dye, aminoFITC, which was also similarly solubilized in the reservoir in which Rhodamine B solution was not injected
(Concentration 10 μM) was injected. Next, drawing alignment was performed using the cross index as a reference. In the drawing alignment, the two cross-shaped indicators are imaged by a CCD camera using laser light or the like as a light source, processed by an image processing device, recognized, and the position coordinates are detected. The XYθ stage that is installed was adjusted to align the head and the carrier. From each nozzle of this liquid discharge head, the solutions of the above-mentioned two kinds of dyes were discharged onto the cleaned glass substrate and applied. Each reservoir and nozzle were washed with the above solvent and each dye solution in advance, and the liquid removal by vacuum suction was appropriately repeated. The amount of one droplet (mainly) is 10 pl, and in this case, the area occupied by the droplets (dots) ejected on the carrier is about 50 μm in diameter, and the interval between dots is about 100 μm.

Nikon fluorescence microscope ECLIPSE E80
0 (Nikon Corporation) 20x objective lens (plan apochromat) and fluorescent filter (B-2A: Rhodamine B)
, B-2E / C: for amino FITC, both are Nikon), and the state of each dye aqueous solution applied was observed with fluorescence at a magnification of 200 times, and it was possible to apply an aqueous solution without color mixing. Met. Further, each dot was arranged in an orderly manner, and no stain or stain derived from mist was observed.

(Example 2) (Evaluation of Color Mixing by Hybridization) The glass substrate was washed in the same manner as in Example 1. Then, the product was purified by vacuum distillation, the following formula (I):

[0138]

Embedded image (CH ₃ O) ₃ SiC ₃ H ₆ NHC ₂ H ₄ NH ₂ (I)

Aminosilane coupling agent (KBM-
603: Compound I, an aqueous solution containing 1% concentration of Shin-Etsu Chemical Co., Ltd.) was stirred at room temperature for 1 hour to hydrolyze the methoxy group portion. Next, after washing the substrate, it is immediately immersed in the aqueous solution of the silane coupling agent, and at room temperature for 1 minute.
Soak for hours. After that, it was washed with running water (ultra pure water), blown with nitrogen gas to be dried, and then heat-fixed in an oven at 120 ° C. for 1 hour.

After cooling, the following formula (II):

[0141]

[Chemical 4]

The substrate was immersed in a 0.3% solution of N- (6-maleimido-caproxy) succinimide (EMCS; compound II) (ethanol: dimethylsulfoxide = 1: 1) at room temperature for 2 hours to obtain EMCS aminosilane. It was reacted with the amino group of the coupling agent. After completion of the reaction, the mixture was washed once with ethanol: dimethyl sulfoxide = 1: 1 and three times with ethanol, and dried by blowing nitrogen gas. 5'-ATGAACCGGAGGCCCATC-3 '3'-TACTTGGCCTCCGGGT-5'5'-AAAAAAAAAAAAAAAAAAAAAAAAA-3'-3'-base sequence complementary to the base sequence of each of the above-mentioned sequences and complementary 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT And having an mercapto group (also referred to as SH group: sulfhydryl group) capable of reacting with the finally purified maleimide group on the surface of the substrate via a linker at the 5'end (compounds III and IV: Bex Co., Ltd.) was used for the oligonucleotide probe used in the verification of this example.
Formula (III, IV):

[0143]

Embedded image _{3'-TACTTGGCCTCCGGGTAG-OP (O 2} ) O- (CH 2) 6 -5 ' Compound _{III 3'-TTTTTTTTTTTTTTTTTTTTTTTTT-OP (O} 2) O - (CH 2) 6 -5' Compound IV

Each of the compounds III and IV was dissolved in the same solvent for ejection as in Example 1 so that the absorbance was 1.0, and every other compound was applied from the liquid ejection head onto the glass substrate in the same manner as in Example 1. . The reservoirs and nozzles were washed with the solution of the solvent and the oligonucleotide in advance and the liquid was removed by vacuum suction as appropriate. After that, the oligonucleotide solution was discharged onto the above substrate. Drop volume,
The dot size, dot interval, ejection pattern, ejection frequency, electrification by the ionizer, and electrification of the mist adsorbing portion are the same as in the first embodiment. Further, the above solvent has a high moisturizing property and can prevent the oligonucleotide solution from drying in the reservoir and on the substrate.

The substrates thus ejected with the solutions of the compounds III and IV were reacted in a moisturizing chamber having a humidity of 100% at room temperature for 1 hour and then washed in running water (ultra pure water) for about 30 seconds. Next, the above substrate contains BSA (bovine serum albumin Sigma-Aldrich Japan) at a concentration of 2%.
0 mM phosphate buffer (pH = 7.0, 1M NaCl
Was immersed in the buffer solution) for 1 hour, washed appropriately with the above buffer solution, and stored in this buffer solution.

(Hybridization) Model Target D
Compounds V and VI (purchased from Beck's Co., Ltd.) having the sequence of Example 1 as NA and having tetramethylrhodamine bound to the 5'end were used for hybridization.

As the model target DNA, the formula (V, V
I): (only V is shown, VI has a DNA portion of A25)

[0148]

[Chemical 6]

A DNA molecule of the above, having the sequence of the above,
Compounds V and VI (purchased from Bex Co., Ltd.) having tetramethylrhodamine bound to the 5'end as a fluorescence index were used to carry out a hybridization reaction with the prepared probe on the substrate.

This hybridization reaction was carried out by using a phosphate buffer solution (10 mM phosphate buffer solution, pH = 7.0, 5) containing two substrates, each containing Compound V and VI at a concentration of 5 nM.
2 ml (containing 0 mM NaCl) was used and performed separately in the hybrid pack. The substrate was sealed in a hybrid pack together with the model target DNA solution, heated to 70 ° C. in a thermostat, then cooled to 50 ° C., and left in that state for 10 hours.

Next, the substrate was taken out from the hybrid pack and washed with a hybridization buffer for the purpose of removing unreacted target DNA. After washing, the substrate was placed on a slide glass in a state of being covered with a buffer solution, covered with a cover glass, and irradiated with laser light from the back surface to observe the fluorescence position based on fluorescence from a fluorescence index.
The fluorescence microscope used for this observation was ECLIPSE E
800 (Nikon Corporation) was set with a 20 × objective lens (plan apochromat) and a fluorescent filter (Y-2E / C). Fluorescence was observed only from the portion of each substrate where the probe complementary to the target DNA was to be present.
The dots were arranged in an orderly manner.

(Embodiment 3) In this embodiment, after a partition wall made of a black matrix is formed and then a dry etching treatment, a plasma treatment, and a water treatment are carried out to prepare a probe fixing carrier, evaluation of color mixture and white spots is carried out. I went. Specifically, it carried out as follows.

[Formation of Black Matrix] On a glass substrate (“1737” manufactured by Corning), a black resist containing carbon black (“C manufactured by FUJIFILM Ohlin”
K-A143B resist "), predetermined exposure with a UV exposure device, development with an inorganic alkaline aqueous solution, and post-baking treatment by heating in a clean oven at 220 ° C for 60 minutes to give a film thickness of 2 µm and 100 µm × 200μ
A black matrix pattern (partition wall) having a rectangular opening of m was produced. The length a of the partition wall was 20 μm.

[Evaluation of Surface Roughness] The surface roughness of the glass substrate used for the formation of the black matrix was evaluated as Digital.
Instrument company "NanoScope III
“AAFM Dimension 3000 stage system” was used to measure at an arbitrary position before forming a black matrix. As a result, the average roughness (Ra) of the glass substrate surface was 3 nm.

[Dry Etching Treatment] Plasma treatment was performed on the above glass substrate (black matrix substrate) on which a black matrix was formed under the following conditions using a cathode coupling parallel plate type plasma treatment apparatus.

Gas used: O ₂ gas flow rate: 80 sccm Pressure: 8 Pa RF power: 0.15 KW Processing time: 30 sec

[Plasma Treatment] After completion of the dry etching treatment, plasma treatment was performed on the black matrix substrate in the same apparatus under the following conditions. Gas used: CF ₄ gas flow rate: 330 sccm Pressure: 44 Pa RF power: 1.2 kW Processing time: 130 sec

[Water Treatment] The black matrix substrate after the plasma treatment was subjected to water treatment. Specifically, the black matrix substrate was immersed in an ultrasonic pure water bath. The processing conditions were as follows. Pure water temperature: 50-60 ° C. Ultrasonic frequency: 40 kHz Treatment time: 5 min Drying: Pulling dry, 60 ° C., 5 minutes

[Evaluation of Water Repellency] Using a Kyowa Interface Co., Ltd. automatic liquid crystal glass cleaning / processing inspection apparatus “LCD-400S”, the contact angle to pure water was measured for the black matrix substrate after the water treatment step. It was measured. The surface of the black matrix was measured on a frame having a width of 5 mm provided around the fine pattern, and the surface of the glass substrate was measured further outside of the frame at a place where the black matrix pattern was not provided. .
The contact angle with respect to each pure water was as follows: glass substrate surface: 12 °, black matrix surface: 125 °.

[Supply of Aqueous Solution to Carrier by Inkjet Method] Next, a solvent for ejecting the fluorescent dye rhodamine B by a liquid ejecting head, that is, glycerin 7.5 wt%, urea 7.5 wt%, thiodiglycol. 7.5 wt%, general formula (IV):

[0161]

[Chemical 7]

An aqueous solution containing 1 wt% of acetylene alcohol (for example, trade name: acetylenol EH Kawaken Fine Chemical Co., Ltd.) represented by was dissolved at a concentration of 10 μM. Next, 20 pl of this rhodamine B aqueous solution was supplied to each opening by an inkjet device. Next, from another inkjet head, 10 μM
20 pl of an aqueous solution of amino FITC in Example 1 was supplied to the other first region. Rhodamine B and amino FITC were used because they are water-soluble and the state of the liquid supplied by fluorescence and the state of cross contamination are observed.

The contact angle of the probe solution with respect to the black matrix substrate was measured using an automatic liquid crystal glass cleaning / processing inspection device "LCD-400S" manufactured by Kyowa Interface Co., Ltd. The surface of the black matrix was measured on a frame having a width of 5 mm provided around the fine pattern, and the surface of the glass substrate was measured further outside of the frame at a place where the black matrix pattern was not provided. . The contact angle of each probe solution was 17 ° on the glass substrate surface and 115 ° on the black matrix surface.

The Nikon fluorescence microscope has 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 by fluorescence at 100 times. Each aqueous solution was uniformly supplied in the first region without forming droplets. Also,
The fluorescence of the other dyes was not observed from the first region to which each dye was to be supplied, and it was possible to supply the aqueous solution without cross contamination to each first region. Further, when observing the diameter of the applied droplet with an optical microscope, the solution was sufficiently wet and spread in the opening, it was difficult to find the end of the droplet, and white spots were observed in all probe carriers of this example. There wasn't.

Comparative Example 1 A probe carrier was prepared in the same manner as in Example 3 except that plasma treatment and water treatment were not performed. The contact angle of the black matrix substrate with respect to pure water was 80 ° on the surface of the glass substrate and 80 ° on the surface of the black matrix.

A probe carrier was prepared in the same manner as in Example 1 except that the water treatment was not performed. The contact angle of the black matrix substrate with respect to pure water was 15 ° on the surface of the glass substrate and 130 ° on the surface of the black matrix.

The spreadability of the solution was also evaluated. As a result, the probe solution did not wet and spread with 20 pl of the probe solution, and white spots were observed at all openings in the probe carrier.

Example 4 A probe was prepared in the same manner as in Example 1 except that the same dry etching treatment, plasma treatment and water treatment as in Example 3 were carried out as a pretreatment for applying the probe solution onto the carrier. A carrier was prepared.

Nikon fluorescence microscope ECLIPSE E80
0 (Nikon Corporation) 20x objective lens (plan apochromat) and fluorescent filter (B-2A: Rhodamine B)
, B-2E / C: for amino FITC, both are Nikon), and the state of each dye aqueous solution applied was observed with fluorescence at a magnification of 200 times, and it was possible to apply an aqueous solution without color mixing. Met. In addition, each dot is arranged in an orderly manner, and no twist or dirt derived from mist is observed.Furthermore, the solution is sufficiently wet and spread in the opening, and it is difficult to find the end of the droplet. No white spots were observed in the probe carrier of 1. Therefore, in the probe carrier of the present embodiment, by providing an index, in addition to being able to more accurately apply the probe solution to a desired position when performing drawing alignment with the index as a reference, plasma treatment, Defluorination treatment,
By producing a probe carrier having a partition wall exhibiting high water repellency with respect to an aqueous probe solution and a probe fixing region exhibiting high hydrophilicity by subjecting the carrier to water treatment and water treatment, a slight misalignment of application position occurs. Even if it happens, the probe solution does not mix with the opening (well) of the adjacent partition due to the water repellency of the partition, and white spots do not occur due to wetting and spreading. A high probe carrier could be achieved.

[0170]

According to the present invention, a carrier having an index in the probe non-immobilized region is used, and a solution containing the probe is applied to a specific position on the carrier by using the position of the index as a reference. By fixing, it is possible to manufacture a highly reliable and highly densified probe carrier without a mixture of probe solutions and white spots accurately and quickly. Also,
The position of the target compound specifically bound to the probe immobilized on the probe carrier manufactured by the above-described manufacturing method can be accurately and quickly detected based on the index. Further, a probe carrier having a partition wall exhibiting high water repellency with respect to an aqueous probe solution and a probe fixing region exhibiting high hydrophilicity by subjecting the carrier to a gas removal treatment such as plasma treatment, defluorination treatment, and water treatment. Can be produced, and a mixture of probe solutions and white spots can be more effectively prevented.

[0171]

[Sequence Listing] SEQUENCE LISTING <110> CANON INC. <120> Probe carrier, Method and Apparatus for prod
ucing Probe carrier <130> 4705007 <150> JP2001 / 133697 <151> 2001-04-27 <150> JP2001 / 133698 <151> 2001-04-27 <160> 4 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to be hybridized wi
th the designed oligonucleotide "gatgggcctccggttca
t "<400> 1 atgaaccgga ggcccatc 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide used as a probe to
be stabilized on a carrier <400> 2 gatgggcctc cggttcat 18 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to be hybridized wi
th the designed oligonucleotide "tttttttttttttttt
ttttttttt "<400> 3 aaaaaaaaaa aaaaaaaaaa aaaaa 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide used as a probe to
be stabilized on a surface of a carrier <400> 4 tttttttttt tttttttttt ttttt 25

[Brief description of drawings]

FIG. 1 is a view exemplifying one embodiment of a probe carrier or a probe-immobilizing carrier according to the present invention in which an index is formed.

FIG. 2 is a process chart schematically showing a method for producing a probe carrier according to the present invention.

FIG. 3 is a conceptual diagram of white spots that occur in a method of manufacturing a probe carrier by an inkjet method.

FIG. 4 is a schematic sectional view of an example of a probe carrier that is an embodiment of the probe carrier of the present invention.

FIG. 5 is a schematic sectional view of an example of a probe carrier that is an embodiment of the probe carrier of the present invention.

FIG. 6 is a schematic sectional view of an example of a probe carrier that is an embodiment of the probe carrier of the present invention.

FIG. 7 is a schematic diagram showing an example of the configuration of a plasma generator that can be used in the manufacturing method of the present invention.

FIG. 8 is a schematic diagram showing another configuration of a plasma generator that can be used in the manufacturing method of the present invention.

FIG. 9 is a configuration diagram of a controller of the probe carrier manufacturing apparatus.

[Explanation of symbols]

11 indicators 12 partitions 13 wells 14 chips 15 Chip periphery 21 carrier 22 Partition layer 23 Photoresist mask 24 Probe solution discharge head 25 probe solution 31 Carrier 32 partitions 33 Probe solution 34 White areas 41 Carrier 42 partition 43 Probe solution 51 carrier 52 partition 53 Probe solution 61 carrier 62 bulkhead 63 probe solution 71 Upper electrode 72 Lower electrode 73 Carrier to be treated 74 high frequency power supply 91 Controller 92 Teaching pendant 93 Operation part 94 Display 95 Interface 96 CPU 97 ROM 98 RAM 99 Discharge controller 100 stage controller 101 Probe carrier manufacturing apparatus

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 37/00 102 C12N 15/00 ZNAF (72) Inventor Naoshi Okamoto 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon In-house F-term (reference) 4B024 AA20 CA01 CA09 CA11 HA14 HA19 4B063 QA13 QQ42 QQ52 QR32 QR35 QR55 QR84 QS34

Claims (29)

[Claims] 1. A method for producing a probe carrier in which a plurality of types of probes capable of specifically binding to a target substance are immobilized on different specific positions on the carrier, the specific positions on the carrier being excluded. A step of providing an index on the part, a step of applying a solution containing the probe to the specific position on the carrier with reference to the position of the index, and a step of fixing the probe on the carrier A method for producing a probe carrier, comprising: 2. The method for producing a probe carrier according to claim 1, further comprising the step of forming a partition wall for partitioning the specific position on the carrier. 3. A step of forming a partition wall for partitioning the specific position on the carrier; a plasma irradiation step of irradiating the carrier with plasma in an atmosphere of a gas containing at least fluorine atoms; The method of manufacturing a probe carrier according to claim 1, further comprising the step of removing the gas component attached to the position. 4. The probe carrier manufacturing method according to claim 3, wherein the step of removing the gas component is a water washing step of washing the gas component by bringing water into contact with the surface of the plasma-irradiated carrier. Method. 5. The method of manufacturing a probe carrier according to claim 2, wherein the partition wall is formed by a photolithography method. 6. The photolithography method comprises forming a photosensitive resin layer on the surface of the carrier, exposing the photosensitive resin layer in a pattern corresponding to the partition walls, and developing to form the partition walls. The method for producing a probe carrier according to claim 5, further comprising a step of firing the partition wall prior to the plasma irradiation step. 7. The method for producing a probe carrier according to claim 4, wherein in the washing step, the carrier is brought into contact with water and then dried at 100 ° C. or lower. 8. The method of manufacturing a probe carrier according to claim 2, wherein the index is provided at a predetermined position of the partition wall. 9. The probe according to claim 1, wherein in the step of applying the solution containing the probe to the specific position, the solution is applied by flying in a space. Method for producing carrier. 10. A method for producing a probe-immobilizing carrier for immobilizing a plurality of types of probes capable of specifically binding to a target substance on a carrier at different specific positions, wherein a region is defined on the carrier. A step of forming partition walls for, a plasma irradiation step of irradiating the carrier with plasma under a gas atmosphere containing at least fluorine atoms, and contacting water with the surface of the carrier subjected to the plasma irradiation to cause the gas component And a washing step of rinsing. 11. A probe carrier, characterized in that a solution containing the probe is applied to the probe-immobilizing carrier produced by the method according to claim 10 at the specific position on the carrier to immobilize the probe. Manufacturing method. 12. A probe carrier in which a plurality of types of probes capable of specifically binding to a target substance are immobilized at different specific positions, and an index is provided on a portion of the carrier other than the specific position. A characteristic probe carrier. 13. The probe carrier according to claim 12, wherein the carrier has a partition wall for partitioning the specific position, and the index is at a predetermined position of the partition wall. 14. The probe carrier according to claim 13, wherein the index is a recess provided in the partition wall. 15. The probe carrier according to claim 13, wherein the partition wall has a light-shielding property. 16. The partition wall is formed of a resin composition containing carbon black.
5. The probe carrier according to item 5. 17. The partition wall is water-repellent, the contact angle of the partition wall with respect to pure water is 120 ° or more, the probe fixing region is hydrophilic, and the probe fixing region is pure water. The probe carrier according to any one of claims 13 to 16, wherein the contact angle with respect to is 30 ° or less. 18. The average value a of the length a of the partition cross-section upper portion in the direction parallel to the substrate at a height between 80% and 100% of the height of the partition top from the bottom of the partition cross section and the partition wall. The relation with the length b of the lower bottom in the section of
The probe carrier according to any one of claims 13 to 17, having a shape represented by /b≧0.7. 19. A probe-immobilizing carrier for immobilizing a plurality of types of probes capable of specifically binding to a target substance at different specific positions, wherein an index is provided on a portion of the carrier excluding the specific positions. A carrier for immobilizing a probe, which comprises: 20. The probe fixing carrier according to claim 19, wherein the carrier has a partition wall for partitioning the specific position, and the index is at a predetermined position of the partition wall. 21. The probe fixing carrier according to claim 20, wherein the index is a recess provided in the partition wall. 22. The probe fixing carrier according to claim 20, wherein the partition wall has a light shielding property. 23. The partition wall is formed of a resin composition containing carbon black.
The probe-immobilizing carrier according to item 2. 24. The partition wall is water repellent, the contact angle of the partition wall with respect to pure water is 120 ° or more, the probe fixing region is hydrophilic, and the probe fixing region is pure water. The probe fixing carrier according to any one of claims 20 to 23, wherein a contact angle with respect to is 30 ° or less. 25. The average value a of the lengths in the direction parallel to the substrate at the upper portion of the partition wall section at a height between 80% and 100% of the height of the partition wall from the bottom of the partition wall to the partition wall and the partition wall. The relation with the length b of the lower bottom in the section of
The probe fixing carrier according to any one of claims 20 to 24, which has a shape represented by /b≧0.7. 26. A method for identifying the position of a probe specifically bound to the target substance by applying the target substance to a plurality of types of probes capable of binding to the target substance, which are carried on a carrier. The reference is characterized by specifying the position of the probe specifically bound to the target substance with reference to an index provided at a position different from the specific position where the plurality of types of probes are carried. Method. 27. The indicator and the target substance are labeled with a radioactive label.
The identification method according to item 6. 28. The indicator and the target substance are labeled with a fluorescent label.
The identification method described in. 29. The carrier is a carrier made of a light-transmissive plate, and a partition wall for partitioning a specific position where the probes of the plurality of types are fixed on one surface of the carrier. 29. The identifying method according to claim 28, further comprising: detecting the fluorescence emitted by the radioactive label and penetrating the carrier on the other surface of the carrier.